Corn event MIR162

ABSTRACT

A novel transgenic corn event designated MIR162 is disclosed. The invention relates to nucleic acids that are unique to event MIR162 and to methods for detecting the presence of the MIR162 event based on DNA sequences of the recombinant constructs inserted into the corn genome that resulted in the MIR162 event and of genomic sequences flanking the insertion site. The invention further relates to corn plants comprising the transgenic genotype of MIR162 and to methods for producing a corn plant by crossing a corn plant comprising the MIR162 genotype with itself or another corn variety. Seeds of corn plants comprising the MIR162 genotype are also objects of the present invention. The invention also relates to methods of controlling insects using MIR162 corn plants.

This application is a divisional of U.S. patent application Ser. No.13/534,202, filed Jun. 27, 2012, which is a divisional of U.S. patentapplication Ser. No. 12/301,824, filed Jul. 15, 2009, now U.S. Pat. No.8,232,456, which is a §371 of PCT/US2007/012301, filed May 24, 2007, andpublished Dec. 13, 2007 as WO 2007/142840, which claims priority fromU.S. Provisional Application No. 60/810,499, filed Jun. 3, 2006. Thesedocuments are incorporated herein by reference in their entirety.

BACKGROUND

The present invention relates generally to the field of plant molecularbiology, plant transformation, and plant breeding. More specifically,the invention relates to insect resistant transgenic corn plantscomprising a novel transgenic genotype and to methods of detecting thepresence of nucleic acids that are unique to the transgenic corn plantsin a sample and compositions thereof.

Plant pests are a major factor in the loss of the world's importantagricultural crops. About $8 billion are lost every year in the U.S.alone due to infestations of non-mammalian pests including insects. Inaddition to losses in field crops, insect pests are also a burden tovegetable and fruit growers, to producers of ornamental flowers, and tohome gardeners.

Insect pests are mainly controlled by intensive applications of chemicalpesticides, which are active through inhibition of insect growth,prevention of insect feeding or reproduction, or cause death. Goodinsect control can thus be reached, but these chemicals can sometimesalso affect other, beneficial insects. Another problem resulting fromthe wide use of chemical pesticides is the appearance of resistantinsect varieties. This has been partially alleviated by variousresistance management practices, but there is an increasing need foralternative pest control agents. Biological pest control agents, such asBacillus thuringiensis (Bt) strains expressing pesticidal toxins likeδ-endotoxins, have also been applied to crop plants with satisfactoryresults, offering an alternative or compliment to chemical pesticides.The genes coding for some of these δ-endotoxins have been isolated andtheir expression in heterologous hosts have been shown to provideanother tool for the control of economically important insect pests. Inparticular, the expression of Bt δ-endotoxins has provided efficientprotection against selected insect pests, and transgenic plantsexpressing such toxins have been commercialized, allowing farmers toreduce applications of chemical insect control agents.

Another family of insecticidal proteins produced by Bacillus speciesduring the vegetative stage of growth (vegetative insecticidal proteins(Vip)) has also been identified. U.S. Pat. Nos. 5,877,012, 6,107,279,and 6,137,033, herein incorporated by reference, describe a new class ofinsecticidal proteins called Vip3. Other disclosures, including WO98/18932, WO 98/33991, WO 98/00546, and WO 99/57282, have also nowidentified homologues of the Vip3 class of proteins. Vip3 codingsequences encode approximately 88 kDa proteins that possess insecticidalactivity against a wide spectrum of lepidopteran pests, including, butnot limited to, black cutworm (BCW, Agrotis ipsilon), fall armyworm(FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliothisvirescens), sugarcane borer, (SCB, Diatraea saccharalis), lessercornstalk borer (LCB, Elasmopalpus lignosellus), and corn earworm (CEW,Helicoverpa zea), and when expressed in transgenic plants, for examplecorn (Zea mays), confer protection to the plant from insect feedingdamage.

Present plant transformation methods generally lead to the randomintegration of transgenes like vip3 into a host-plant genome. Thisrandom insertion of introduced DNA into the plant's genome can be lethalif the foreign DNA happens to insert into, and thus mutate, a criticallyimportant native gene. In addition, even if a random insertion eventdoes not impair the functioning of a host cell gene, the expression ofan inserted foreign gene may be influenced by “position effects” causedby the surrounding genomic DNA. In some cases, the gene is inserted intosites where the position effects are strong enough to prevent thesynthesis of an effective amount of product from the introduced gene.For example, it has been observed in plants that there may be widevariations in levels of expression of a heterologous gene introducedinto a plant's chromosome among individually selected events. There mayalso be differences in spatial or temporal patterns of expression, forexample, differences in the relative expression of a transgene invarious plant tissues, that may not correspond to the patterns expectedfrom transcriptional regulatory elements present in the introduced geneconstruct. In other instances, overproduction of the gene product hasdeleterious effects on the cell. Because of these potential problems, itis common to produce hundreds of different events and screen thoseevents for a single event that has desired transgene expression patternsand levels for commercial purposes. However, once a commercially viablesite within the plant's genome is identified it would be advantageous totarget genes of interest to that non-detrimental site.

Several methods for the targeted insertion of a nucleotide sequence ofinterest into a specific chromosomal site within a plant cell have beendescribed. Site-specific recombination systems have been identified inseveral prokaryotic and lower eukaryotic organisms. Such systemstypically comprise one or more proteins that recognize two copies of aspecific nucleotide sequence, cleave and ligate those nucleotidesequences, and thereby provide a precise, site-specific exchange ofgenetic information. Several site-specific recombinases are known in theart. These include, but are not limited to, e.g., the bacteriophage P1Cre/lox system (Austin et al. (1981) Cell 25: 729-736), the R/RSrecombinase system from the pSRi plasmid of the yeast Zygosaccharomycesrouxii (Araki et al. (1985) J. Mol. Biol. 182: 191-203), the Gin/gixsystem of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230:170-176), the FLP/FRT recombinase system from the 2 .mu.m plasmid of theyeast Saccharomyces cerevisiae (Broach et al. (1982) Cell 29: 227-234),and the Int recombinase from bacteriophage Lambda (Landy (1989) Annu.Rev. Biochem. 58: 912-949; Landy (1993) Curr. Opin. Genet. Dev. 3:699-707; Lorbach et al. (2000) J. Mol. Biol. 296: 1175-1181; and WO01/16345). One particularly useful site-specific targeting approach,disclosed in US Patent Application Publication No. 2006/0130179, hereinincorporated by reference, uses lambda integrase mediated recombination.The method comprises introducing into a plant cell a target nucleotidesequence comprising a first Integrase Recognition Site; introducing intothe plant cell a donor nucleotide sequence comprising a second IntegraseRecognition Site; and introducing into the plant cell an Integrase orIntegrase complex. Another useful site-specific targeting approach isdisclosed in US Patent Application Publication No. 2006/0253918, hereinincorporated by reference, which uses homologous recombination tointegrate one or more genes (gene stacking) at specific locations in thegenome.

An event that has desired levels or patterns of transgene expression isuseful for introgressing the transgene into other genetic backgrounds bysexual out-crossing using conventional breeding methods. Progeny of suchcrosses maintain the transgene expression characteristics of theoriginal transformant. This strategy is used to ensure reliable geneexpression in a number of varieties that are well adapted to localgrowing conditions. It would also be advantageous to be able to detectthe presence of a particular event in order to determine whether progenyof a sexual cross contain a transgene of interest. In addition, a methodfor detecting a particular event would be helpful for complying withregulations requiring the pre-market approval and labeling of foodsderived from recombinant crop plants, for example. It is possible todetect the presence of a transgene by any well-known nucleic aciddetection method including but not limited to thermal amplification(polymerase chain reaction (PCR)) using polynucleotide primers or DNAhybridization using nucleic acid probes. Typically, for the sake ofsimplicity and uniformity of reagents and methodologies for use indetecting a particular DNA construct that has been used for transformingvarious plant varieties, these detection methods generally focus onfrequently used genetic elements, for example, promoters, terminators,and marker genes, because for many DNA constructs, the coding sequenceregion is interchangeable. As a result, such methods may not be usefulfor discriminating between constructs that differ only with reference tothe coding sequence. In addition, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNAadjacent to the inserted heterologous DNA (“flanking DNA”) is known.

For the foregoing reasons, there is a need for insect resistanttransgenic corn events comprising novel nucleic acid sequences which areunique to the transgenic corn event, useful for identifying thetransgenic corn event and for detecting nucleic acids from thetransgenic corn event in a biological sample, as well as kits comprisingthe reagents necessary for use in detecting these nucleic acids in abiological sample. There is a further need to provide specific targetsites within the maize genome to allow for targeting and control ofinsertion of nucleotide sequences to be integrated into the corn genome.

SUMMARY

The present invention relates to a transformed corn (Zea mays) event,designated MIR162 comprising a novel transgenic genotype that comprisesa vip3Aa20 coding sequence, which is unique to event MIR162. Thevip3Aa20 coding sequence encodes a Vip3Aa20 insecticidal protein thatconfers insect resistance to MIR162 corn plants. The MIR162 event alsocomprises a pmi coding sequence encoding a PMI protein that confers uponcorn cells the ability to utilize mannose as a carbon source. Inaddition to the vip3A20 coding sequence, the present invention alsoprovides other nucleic acids that are unique to MIR162. The inventionalso provides transgenic corn plants comprising the nucleic acids uniqueto MIR162, seed from the transgenic corn plants, and to methods forproducing a transgenic corn plant comprising the unique nucleic acids ofthe invention by crossing a corn inbred comprising the nucleic acidsunique to MIR162 with itself or another corn line of a differentgenotype. An example of seed, and hence corn plants grown from the seed,comprising nucleic acids unique to MIR162 was deposited at the AmericanType Culture Collection as accession No. PTA-8166. The transgenic cornplants of the invention may have essentially all of the morphologicaland physiological characteristics of corresponding isogenicnon-transgenic corn plants in addition to those conferred upon the cornplants by the novel genotype of the invention. Biological samples andextracts from MIR162 corn plants, tissues and seeds are also provided bythe present invention. The present invention also provides compositionsand methods for detecting the presence of nucleic acids unique to MIR162in biological samples based on the DNA sequence of the recombinantexpression cassettes inserted into the corn genome that resulted in theMIR162 event and of genomic sequences flanking the insertion site. Thepresent invention also provides a non-detrimental insertion target siteon a maize chromosome useful for inserting genes of interest to aspecific location on the chromosome and to methods of altering a maizegenome by inserting heterologous nucleic acids at the disclosedinsertion site or in the vicinity of the disclosed insertion site. TheMIR162 event can be further characterized by analyzing expression levelsof the Vip3Aa20 and PMI proteins as well as by testing MIR162 forefficacy against lepidopteran insect pests. The present invention alsoprovides methods of producing transgenic corn plants resistant to abroader spectrum of insect pests by stacking the Vip3Aa20 insectresistant trait with insect resistance traits different than Vip3Aa20.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the Vip3Aa20 coding sequence in MIR162.

SEQ ID NO: 2 is the Vip3Aa20 amino acid sequence.

SEQ ID NO: 3 is the sequence of plasmid pNOV1300.

SEQ ID Nos: 4-12 are primers and probes useful in a TAQMAN assay.

SEQ ID NO: 13 is the sequence of a vip3Aa20 probe.

SEQ ID NO: 14 is the sequence of a pmi probe.

SEQ ID Nos: 15-37 are primers useful in the present invention.

SEQ ID No: 38 is the sequence of a vip3Aa20 amplicon.

SEQ ID Nos: 39-40 are primers useful in the present invention.

SEQ ID No: 41 is the sequence of the CJ134/179 5′ amplicon.

SEQ ID Nos: 42-43 are primers useful in the present invention.

SEQ ID NO: 44 is a vip3Aa20 3′ amplicon.

SEQ ID NO: 45 is the 5′ genome-insert junction.

SEQ ID NO: 46 is corn genome sequence flanking 5′ to insert.

SEQ ID NO: 47 is the 3′ insert-genome junction.

SEQ ID NO: 48 is corn genome flanking 3′ to insert.

SEQ ID NO: 49 is the MIR162 insert and flanking sequences.

SEQ ID Nos. 50-54 are primers useful in the present invention.

SEQ ID NO: 55 is a 5′ PCR amplicon

SEQ ID Nos. 56-58 are primers useful in the present invention.

SEQ ID NO: 59 is a 3′ PCR amplicon.

SEQ ID Nos. 60-105 are primers useful in the present invention.

SEQ ID NO: 106 is the sequence of the region of maize chromosome 5comprising the disclosed chromosomal target site.

SEQ ID NO: 107 is the maize genomic sequence that was displaced by theinsertion of heterologous DNA in MIR162.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms usedherein are to be understood according to conventional usage by those ofordinary skill in the relevant art. Definitions of common terms inmolecular biology may also be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5^(th) edition, Springer-Verlag: NewYork, 1994. The nomenclature for DNA bases and amino acids as set forthin 37 C.F.R. §1.822 is used herein.

As used herein, the term “amplified” means the construction of multiplecopies of a nucleic acid molecule or multiple copies complementary tothe nucleic acid molecule using at least one of the nucleic acidmolecules as a template. Amplification systems include, but not limitedto the polymerase chain reaction (PCR) system, ligase chain reaction(LCR) system, nucleic acid sequence based amplification (NASBA, Cangene,Mississauga, Ontario), Q-Beta Replicase systems, transcription-basedamplification system (TAS), and strand displacement amplification (SDA).See, e.g., Diagnostic Molecular Microbiology Principles andApplications, D. H. Persing et al., Ed., American Society forMicrobiology, Washington, D.C. (1993). The product of amplification istermed an amplicon.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Preferably the RNA is then translated in an organism to produce aprotein.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

“Detection kit” as used herein refers to a kit of parts useful indetecting the presence or absence of DNA unique to MIR162 plants in asample, wherein the kit comprises nucleic acid probes and/or primers ofthe present invention, which hybridize specifically under highstringency conditions to a target DNA sequence, and other materialsnecessary to enable nucleic acid hybridization or amplification methods.

As used herein the term transgenic “event” refers to a recombinant plantproduced by transformation and regeneration of a plant cell or tissuewith heterologous DNA, for example, an expression cassette that includesa gene of interest. The term “event” refers to the original transformantand/or progeny of the transformant that include the heterologous DNA.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another corn line. Even after repeatedbackcrossing to a recurrent parent, the inserted DNA and the flankingDNA from the transformed parent is present in the progeny of the crossat the same chromosomal location. The term “event” also refers to DNAfrom the original transformant comprising the inserted DNA and flankinggenomic sequence immediately adjacent to the inserted DNA that would beexpected to be transferred to a progeny that receives inserted DNAincluding the transgene of interest as the result of a sexual cross ofone parental line that includes the inserted DNA (e.g., the originaltransformant and progeny resulting from selfing) and a parental linethat does not contain the inserted DNA. Normally, transformation ofplant tissue produces multiple events, each of which represent insertionof a DNA construct into a different location in the genome of a plantcell. Based on the expression of the transgene or other desirablecharacteristics, a particular event is selected. Thus, “event MIR162”,“MIR162” or “MIR162 event” may be used interchangeably.

An insect resistant MIR162 corn plant can be bred by first sexuallycrossing a first parental corn plant consisting of a corn plant grownfrom a transgenic MIR162 corn plant, such as a MIR162 corn plant grownfrom the seed deposited at the ATCC under accession No. PTA-6188, andprogeny thereof derived from transformation with the expressioncassettes of the embodiments of the present invention that confersinsect resistance, and a second parental corn plant that lacks insectresistance, thereby producing a plurality of first progeny plants; andthen selecting a first progeny plant that is resistant to insects; andselfing the first progeny plant, thereby producing a plurality of secondprogeny plants; and then selecting from the second progeny plants aninsect resistant plant. These steps can further include theback-crossing of the first insect resistant progeny plant or the secondinsect resistant progeny plant to the second parental corn plant or athird parental corn plant, thereby producing a corn plant that isresistant to insects.

“Expression cassette” as used herein means a nucleic acid moleculecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassette may alsocomprise sequences not necessary in the direct expression of anucleotide sequence of interest but which are present due to convenientrestriction sites for removal of the cassette from an expression vector.The expression cassette comprising the nucleotide sequence of interestmay be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.Typically, however, the expression cassette is heterologous with respectto the host, i.e., the particular nucleic acid sequence of theexpression cassette does not occur naturally in the host cell and musthave been introduced into the host cell or an ancestor of the host cellby a transformation process known in the art. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter that initiatestranscription only when the host cell is exposed to some particularexternal stimulus. In the case of a multicellular organism, such as aplant, the promoter can also be specific to a particular tissue, ororgan, or stage of development. An expression cassette, or fragmentthereof, can also be referred to as “inserted sequence” or “insertionsequence” when transformed into a plant.

A “gene” is a defined region that is located within a genome and that,besides the aforementioned coding sequence, may comprise other,primarily regulatory, nucleic acid sequences responsible for the controlof the expression, that is to say the transcription and translation, ofthe coding portion. A gene may also comprise other 5′ and 3′untranslated sequences and termination sequences. Further elements thatmay be present are, for example, introns.

“Gene of interest” refers to any gene which, when transferred to aplant, confers upon the plant a desired characteristic such asantibiotic resistance, virus resistance, insect resistance, diseaseresistance, or resistance to other pests, herbicide tolerance, improvednutritional value, improved performance in an industrial process oraltered reproductive capability.

“Genotype” as used herein is the genetic material inherited from parentcorn plants not all of which is necessarily expressed in the descendantcorn plants. The MIR162 genotype refers to the heterologous geneticmaterial transformed into the genome of a plant as well as the geneticmaterial flanking the inserted sequence.

A “heterologous” nucleic acid sequence is a nucleic acid sequence notnaturally associated with a host cell into which it is introduced,including non-naturally occurring multiple copies of a naturallyoccurring nucleic acid sequence.

A “homologous” nucleic acid sequence is a nucleic acid sequencenaturally associated with a host cell into which it is introduced.

“Operably-linked” refers to the association of nucleic acid sequences ona single nucleic acid fragment so that the function of one affects thefunction of the other. For example, a promoter is operably-linked with acoding sequence or functional RNA when it is capable of affecting theexpression of that coding sequence or functional RNA (i.e., that thecoding sequence or functional RNA is under the transcriptional controlof the promoter). Coding sequences in sense or antisense orientation canbe operably-linked to regulatory sequences.

“Primers” as used herein are isolated nucleic acids that are annealed toa complimentary target DNA strand by nucleic acid hybridization to forma hybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a polymerase, such as DNA polymerase.Primer pairs or sets can be used for amplification of a nucleic acidmolecule, for example, by the polymerase chain reaction (PCR) or otherconventional nucleic-acid amplification methods.

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, such as aradioactive isotope, ligand, chemiluminescent agent, or enzyme. Such aprobe is complimentary to a strand of a target nucleic acid, in the caseof the present invention, to a strand of genomic DNA from corn eventMIR162. The DNA of MIR162 can be from a corn plant or from a sample thatincludes DNA from MIR162. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and can be used to detect the presence of that target DNAsequence.

Primers and probes are generally between 10 and 15 nucleotides or morein length. Primers and probes can also be at least 20 nucleotides ormore in length, or at least 25 nucleotides or more, or at least 30nucleotides or more in length. Such primers and probes hybridizespecifically to a target sequence under high stringency hybridizationconditions. Primers and probes according to the present invention mayhave complete sequence complementarity with the target sequence,although probes differing from the target sequence and which retain theability to hybridize to target sequences may be designed by conventionalmethods.

As used herein gene or trait “stacking” is combining desired traits intoone transgenic line. Plant breeders stack transgenic traits by makingcrosses between parents that each have a desired trait and thenidentifying offspring that have both of these desired traits. Anotherway to stack genes is by transferring two or more genes into the cellnucleus of a plant at the same time during transformation. Another wayto stack genes is by re-transforming a transgenic plant with anothergene of interest. For example, gene stacking can be used to combine twodifferent insect resistance traits, an insect resistance trait and adisease resistance trait, or a herbicide resistance trait. The use of aselectable marker in addition to a gene of interest would also beconsidered gene stacking.

“Stringent conditions” or “stringent hybridization conditions” includereference to conditions under which a probe will hybridize to its targetsequence, to a detectably greater degree than to other sequences.Stringent conditions are target-sequence-dependent and will differdepending on the structure of the polynucleotide. By controlling thestringency of the hybridization and/or wash conditions, target sequencescan be identified which are 100% complementary to the probe (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Longer sequences hybridize specificallyat higher temperatures. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, Part I, Chapter 2 “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier: New York; andCurrent Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds.,Greene Publishing and Wiley-Interscience: New York (1995), and alsoSambrook et al. (2001) Molecular Cloning: A Laboratory Manual (5^(th)Ed. Cols Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. Generally, high stringency hybridization and washconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength andpH. The T_(m) is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. Typically, under high stringency conditions a probe willhybridize to its target subsequence, but to no other sequences.

An example of high stringency hybridization conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of very high stringency wash conditions is 0.15MNaCl at 72° C. for about 15 minutes. An example of high stringency washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook,infra, for a description of SSC buffer).

Exemplary hybridization conditions for the present invention includehybridization in 7% SDS, 0.25 M NaPO₄ pH 7.2 at 67° C. overnight,followed by two washings in 5% SDS, 0.20 M NaPO₄ pH7.2 at 65° C. for 30minutes each wash, and two washings in 1% SDS, 0.20 M NaPO₄ pH7.2 at 65°C. for 30 minutes each wash. An exemplary medium stringency wash for aduplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15minutes. An exemplary low stringency wash for a duplex of, e.g., morethan 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes.

For probes of about 10 to 50 nucleotides, high stringency conditionstypically involve salt concentrations of less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.High stringency conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other underhigh stringency conditions are still substantially identical if theproteins that they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

The following are exemplary sets of hybridization/wash conditions thatmay be used to hybridize nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the present invention: areference nucleotide sequence preferably hybridizes to the referencenucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., moredesirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably stillin 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C.with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C. The sequences of the present invention may bedetected using all the above conditions. For the purposes of definingthe invention, the high stringency conditions are used.

“Transformation” is a process for introducing heterologous nucleic acidinto a host cell or organism. In particular, “transformation” means thestable integration of a DNA molecule into the genome of an organism ofinterest.

“Transformed/transgenic/recombinant” refer to a host organism such as abacterium or a plant into which a heterologous nucleic acid molecule hasbeen introduced. The nucleic acid molecule can be stably integrated intothe genome of the host or the nucleic acid molecule can also be presentas an extrachromosomal molecule. Such an extrachromosomal molecule canbe auto-replicating. Transformed cells, tissues, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof. A “non-transformed”,“non-transgenic”, or “non-recombinant” host refers to a wild-typeorganism, e.g., a bacterium or plant, which does not contain theheterologous nucleic acid molecule. As used herein, “transgenic” refersto a plant, plant cell, or multitude of structured or unstructured plantcells having integrated, via well known techniques of geneticmanipulation and gene insertion, a sequence of nucleic acid representinga gene of interest into the plant genome, and typically into achromosome of a cell nucleus, mitochondria or other organelle containingchromosomes, at a locus different to, or in a number of copies greaterthan, that normally present in the native plant or plant cell.Transgenic plants result from the manipulation and insertion of suchnucleic acid sequences, as opposed to naturally occurring mutations, toproduce a non-naturally occurring plant or a plant with a non-naturallyoccurring genotype. Techniques for transformation of plants and plantcells are well known in the art and may comprise for exampleelectroporation, microinjection, Agrobacterium-mediated transformation,and ballistic transformation.

As used herein, the term “unique” to MIR162 means distinctivelycharacteristic of MIR162. Therefore, nucleic acids unique to eventMIR162 are not found in other non-MIR162 corn plants.

The “Vip3” class of proteins comprises, for example, Vip3Aa, Vip3Ab,Vip3Ac, Vip3Ad, Vip3Ae, VipAf, Vip3Ag, Vip3Ba, and Vip3Bb, and theirhomologues. “Homologue” means that the indicated protein or polypeptidebears a defined relationship to other members of the Vip3 class ofproteins. “Vip3Aa20” is a Vip3 homologue unique to event MIR162. It wasgenerated by spontaneous mutations introduced into the maize-optimizedvip3Aa19 gene comprised in pNOV1300 (SEQ ID NO: 3) during the planttransformation process.

This invention relates to a genetically improved line of corn thatproduces an insect control protein, Vip3Aa20, and a phosphomannoseisomerase enzyme (PMI) that allows the plant to utilize mannose as acarbon source. The invention is particularly drawn to a transgenic cornevent designated MIR162 comprising a novel genotype, as well as tocompositions and methods for detecting nucleic acids unique to MIR162 ina biological sample. The invention is further drawn to corn plantscomprising the MIR162 genotype, to transgenic seed from the corn plants,and to methods for producing a corn plant comprising the MIR162 genotypeby crossing a corn inbred comprising the MIR162 genotype with itself oranother corn line. Corn plants comprising the MIR162 genotype of theinvention are useful in controlling lepidopteran insect pests including,but not limited to, black cutworm (BCW, Agrotis ipsilon), fall armyworm(FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliothisvirescens), sugarcane borer (SCB, Diatraea saccharalis), lessercornstalk borer (LCB, Elasmopalpus lignosellus), corn earworm (CEW,Helicoverpa zea), and western bean cutworm (WBCW, Striacosta albicosta).The invention is further drawn to a method of protecting transgenic cornfrom feeding damage whereby stacking the insect resistance trait ofMIR162 with a different insect resistance trait in the same transgenicplant results is a corn plant that is protected from feeding damage to agreater degree than the insect resistance traits alone.

In one embodiment, the present invention encompasses an isolated nucleicacid molecule comprising a nucleotide sequence that is unique to eventMIR162.

In another embodiment, the present invention encompasses an isolatednucleic acid molecule that links a heterologous DNA molecule insertedinto the genome of MIR162 to genome DNA in MIR162 comprising at least 10or more (for example 15, 20, 25, 50 or more) contiguous nucleotides ofthe heterologous DNA molecule and at least 10 or more (for example 15,20, 25, 50, or more) contiguous nucleotides of the genome DNA flankingthe point of insertion of the heterologous DNA molecule. Also includedare nucleotide sequences that comprise 10 or more nucleotides ofcontiguous insert sequence from event MIR162 and at least one nucleotideof flanking DNA from event MIR162 adjacent to the insert sequence. Suchnucleotide sequences are unique to and diagnostic for event MIR162.Nucleic acid amplification or hybridization of genomic DNA from MIR162produces an amplicon comprising such unique sequences and is diagnosticfor event MIR162. In one aspect of this embodiment, the nucleotidesequence is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQID NO: 55, SEQ ID NO: 59, and the complements thereof.

In another embodiment, the invention encompasses an isolated nucleicacid molecule comprising a nucleotide sequence which comprises at leastone junction sequence of event MIR162, wherein a junction sequence spansthe junction between a heterologous expression cassette inserted intothe corn genome and DNA from the corn genome flanking the insertion siteand is diagnostic for the event. In one aspect of this embodiment, thejunction sequence is selected from the group consisting of SEQ ID NO:45, SEQ ID NO: 47, and the complements thereof.

In another embodiment, the present invention encompasses an isolatednucleic acid molecule linking a heterologous DNA molecule to the cornplant genome in event MIR162 comprising a sequence of from about 11 toabout 20 contiguous nucleotides selected from the group consisting ofSEQ ID NO: 45, SEQ ID NO: 47, and the complements thereof.

In another embodiment, the invention encompasses an isolated nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and thecomplements thereof. In one aspect of this embodiment, the isolatednucleic acid molecule is comprised in a corn seed deposited at theAmerican Type Culture Collection under the accession No. PTA-8166, or inplants grown from the seed.

In one embodiment of the present invention, an amplicon comprising anucleotide sequence unique to event MIR162 is provided. In one aspect ofthis embodiment, the nucleotide sequence is selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and thecomplements thereof.

In another embodiment, the present invention encompasses flankingsequence primers for detecting event MIR162. Such flanking sequenceprimers comprise a nucleotide sequence of at least 10-15 contiguousnucleotides from the 5′ or the 3′ flanking sequence. In one aspect ofthis embodiment, the contiguous nucleotides are selected fromnucleotides 1-1088 (inclusive) of SEQ ID NO: 49 (arbitrarily designatedherein as the 5′ flanking sequence), or the complements thereof. Inanother aspect of this embodiment, the 5′ flanking sequence primers areselected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 39, SEQID NO: 53, SEQ ID NOs: 68-80, and the complements thereof. In anotheraspect of this embodiment, the contiguous nucleotides are selected fromnucleotides 9391-10579 (inclusive) of SEQ ID NO: 49 (arbitrarilydesignated herein as the 3′ flanking sequence), or the complementsthereof. In yet another aspect of this embodiment, the 3′ flankingsequence primers are selected from the group consisting of SEQ ID NO:58, SEQ ID NOs: 97-105, and the complements thereof.

In still another embodiment, the present invention encompasses a pair ofpolynucleotide primers comprising a first polynucleotide primer and asecond polynucleotide primer that function together in the presence of aevent MIR162 DNA template in a sample to produce an amplicon diagnosticfor event MIR162. In one aspect of this embodiment, the first primerand/or the second primer is chosen from SEQ ID NO: 1 or the complimentthereof. In another aspect of this embodiment, the first primer and/orthe second primer is selected from the group consisting of SEQ ID NOs:15-35, SEQ ID NO: 37, SEQ ID NO: 42, and the complements thereof. In yetanother aspect of this embodiment, the amplicon that is produced by thepair of primers comprises SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 44, orthe complements thereof.

In another embodiment, the present invention encompasses a pair ofpolynucleotide primers comprising a first polynucleotide primer and asecond polynucleotide primer which function together in the presence ofa event MIR162 DNA template in a sample to produce an amplicondiagnostic for event MIR162, wherein the first primer is or iscomplementary to a corn plant genome sequence flanking the point ofinsertion of a heterologous DNA sequence inserted into the genome ofevent MIR162, and the second polynucleotide primer sequence is or iscomplementary to the heterologous DNA sequence inserted into the genomeof event MIR162.

In one aspect of this embodiment, the first polynucleotide primercomprises at least 10 contiguous nucleotides from a nucleotide sequenceselected from the group consisting of nucleotides 1-1088 of SEQ ID NO:49, nucleotides 9391-10579 of SEQ ID NO: 49, and the complementsthereof. In a further aspect of this embodiment, the first primer isselected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 39, SEQID NO: 53, SEQ ID NO: 57, SEQ ID NOs: 68-72, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NOs: 97-105, and the complements thereof. In another aspectof this embodiment, the second polynucleotide primer comprises at least10 contiguous nucleotides from position 1089-9390 of SEQ ID NO: 49, orcomplements thereof. In still a further aspect of this embodiment, thesecond polynucleotide primer is selected from the group consisting ofSEQ ID NOs: 15-35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NOs: 50-52, SEQID NOs: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 63, SEQ ID NO: 73,SEQ ID NO: 82, SEQ ID NO: 96, and the complements thereof.

In another aspect of this embodiment, the first polynucleotide primer,which is set forth in SEQ ID NO: 36, and the second polynucleotideprimer which is set forth in SEQ ID NO: 37, function together in thepresence of a event MIR162 DNA template in a sample to produce anamplicon diagnostic for event MIR162 as described in Example 4. In oneembodiment of this aspect, the amplicon comprises the nucleotidesequence set forth in SEQ ID NO: 38.

In yet another aspect of this embodiment, the first polynucleotideprimer, which is set forth in SEQ ID NO: 39, and the secondpolynucleotide primer, which is set forth in SEQ ID NO: 40, functiontogether in the presence of a corn event MIR162 DNA template in a sampleto produce an amplicon diagnostic for the corn event MIR162 as describedin Example 4. In one embodiment of this aspect, the amplicon comprisesthe nucleotide sequence set forth in SEQ ID NO: 41.

In another aspect of this embodiment, the first polynucleotide primer,which is set forth in SEQ ID NO: 53, and the second polynucleotideprimer, which is set forth in SEQ ID NO: 54, function together in thepresence of a corn event MIR162 DNA template in a sample to produce anamplicon diagnostic for the corn event MIR162 as described in Example 5.In one embodiment, the amplicon comprises the nucleotide sequence setforth in SEQ ID NO: 55.

In a still a further aspect of this embodiment, the first polynucleotideprimer, which is set forth in SEQ ID NO: 58, and the secondpolynucleotide primer, which is set forth in SEQ ID NO: 56, functiontogether in the presence of a corn event MIR162 DNA template in a sampleto produce an amplicon diagnostic for the corn event MIR162 as describedin Example 5. In one embodiment, the amplicon comprises the nucleotidesequence set forth in SEQ ID NO: 59.

Of course, it is well within the skill in the art to obtain additionalsequence further out into the genome sequence flanking either end of theinserted heterologous DNA sequences for use as a primer sequence thatcan be used in such primer pairs for amplifying the sequences that arediagnostic for the MIR162 event. For the purposes of this disclosure,the phrase “further out into the genome sequence flanking either end ofthe inserted heterologous DNA sequences” refers specifically to asequential movement away from the ends of the inserted heterologous DNAsequences, the points at which the inserted DNA sequences are adjacentto native genomic DNA sequence, and out into the genomic DNA of theparticular chromosome into which the heterologous DNA sequences wereinserted. Preferably, a primer sequence corresponding to orcomplementary to a part of the insert sequence should prime thetranscriptional extension of a nascent strand of DNA or RNA toward thenearest flanking sequence junction. Consequently, a primer sequencecorresponding to or complementary to a part of the genomic flankingsequence should prime the transcriptional extension of a nascent strandof DNA or RNA toward the nearest flanking sequence junction. A primersequence can be, or can be complementary to, a heterologous DNA sequenceinserted into the chromosome of the plant, or a genomic flankingsequence. One skilled in the art would readily recognize the benefit ofwhether a primer sequence would need to be, or would need to becomplementary to, the sequence as set forth within the insertedheterologous DNA sequence or as set forth SEQ ID NO: 38 depending uponthe nature of the product desired to be obtained through the use of thenested set of primers intended for use in amplifying a particularflanking sequence containing the junction between the genomic DNAsequence and the inserted heterologous DNA sequence.

In another embodiment, the present invention encompasses an isolatedinsecticidal protein comprising SEQ ID NO: 2 and a nucleic acid moleculeencoding SEQ ID NO: 2. In one aspect of this embodiment, the nucleicacid molecule is SEQ ID NO: 1. The present invention also encompasses achimeric gene comprising a heterologous promoter operably linked to thenucleic acid molecule, and to recombinant vectors and host cellscomprising the chimeric gene.

In yet another embodiment, the present invention encompasses a method ofdetecting the presence of a nucleic acid molecule that is unique toevent MIR162 in a sample comprising corn nucleic acids, wherein themethod comprises: (a) contacting the sample with a pair ofpolynucleotide primers that, when used in a nucleic acid amplificationreaction with genomic DNA from event MIR162 produces an amplicon that isdiagnostic for event MIR162; (b) performing a nucleic acid amplificationreaction, thereby producing the amplicon; and (c) detecting theamplicon. In one aspect of this embodiment, the amplicon comprises anucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:49, SEQ ID NO: 55, SEQ ID NO: 59, and the compliments thereof.

In another embodiment, the present invention encompasses a method ofdetecting the presence of a nucleic acid molecule that is unique toevent MIR162 in a sample comprising corn nucleic acids, wherein themethod comprises: (a) contacting the sample with a probe that hybridizesunder high stringency conditions with genomic DNA from event MIR162 anddoes not hybridize under high stringency conditions with DNA from acontrol corn plant; (b) subjecting the sample and probe to highstringency hybridization conditions; and (c) detecting hybridization ofthe probe to the DNA. Detection can be by any means well known in theart including fluorescent, chemiluminescent, radiological,immunological, and the like. In the case in which hybridization isintended to be used as a means for amplification of a particularsequence to produce an amplicon which is diagnostic for the MIR162event, the production and detection by any means well known in the artof the amplicon is intended to be indicative of the intendedhybridization to the target sequence where one probe or primer isutilized, or sequences where two or more probes or primers are utilized.The term “biological sample” is intended to comprise a sample thatcontains or is suspected of containing a nucleic acid comprising frombetween five and ten nucleotides either side of the point at which oneor the other of the two terminal ends of the inserted heterologous DNAsequence contacts the genomic DNA sequence within the chromosome intowhich the heterologous DNA sequence was inserted, herein also known asthe junction sequences. In addition, the junction sequence comprises aslittle as two nucleotides: those being the first nucleotide within theflanking genomic DNA adjacent to and covalently linked to the firstnucleotide within the inserted heterologous DNA sequence. In one aspectof this embodiment, the probe comprises a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO:59, and the complements thereof.

In yet another embodiment, the present invention encompasses a kit forthe detection of nucleic acids that are unique to event MIR162 inbiological sample. The kit includes at least one nucleic acid moleculeof sufficient length of contiguous polynucleotides to function as aprimer or probe in a nucleic acid detection method, and which uponamplification of or hybridization to a target nucleic acid sequence in asample followed by detection of the amplicon or hybridization to thetarget sequence, are diagnostic for the presence of nucleic acidsequences unique to event MIR162 in the sample. The kit further includesother materials necessary to enable nucleic acid hybridization oramplification methods. In one aspect of this embodiment, a nucleic acidmolecule contained in the kit comprises a nucleotide sequence from SEQID NO: 1 or SEQ ID NO: 49. In another aspect of this embodiment, thenucleic acid molecule is a primer selected from the group consisting ofSEQ ID NOs: 15-37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ IDNO: 43, SEQ ID NOs: 50-54, SEQ ID NOs: 56-58, SEQ ID NOs: 60-105, andthe complements thereof. In yet another aspect of this embodiment, theamplicon comprises SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, orthe complements thereof. A variety of detection methods can be usedincluding, but not limited to TAQMAN (Perkin Elmer), thermalamplification, ligase chain reaction, southern hybridization, ELISAmethods, and colorimetric and fluorescent detection methods. Inparticular the present invention provides for kits for detecting thepresence of the target sequence, i.e., at least the vip3Aa20 sequence ora junction sequence, in a sample containing genomic nucleic acid fromMIR162. The kit is comprised of at least one polynucleotide capable ofbinding to the target site or substantially adjacent to the target siteand at least one means for detecting the binding of the polynucleotideto the target site. The detecting means can be fluorescent,chemiluminescent, colorimetric, or isotopic and can be coupled at leastwith immunological methods for detecting the binding. A kit is alsoenvisioned which can detect the presence of the target site in a sample,i.e., at least the vip3Aa20 sequence or a junction sequence of MIR162,taking advantage of two or more polynucleotide sequences which togetherare capable of binding to nucleotide sequences adjacent to or withinabout 100 base pairs, or within about 200 base pairs, or within about500 base pairs or within about 1000 base pairs of the target sequenceand which can be extended toward each other to form an amplicon whichcontains at least the target site.

In another embodiment, the present invention encompasses a method ofdetecting Vip3Aa20 protein in a biological sample, the methodcomprising: (a) extracting protein from event MIR162 tissue; (b)assaying the extracted protein using an immunological method comprisingantibody specific for the Vip3Aa20 protein produced by the MIR162 event;and (c) detecting the binding of said antibody to the Vip3Aa20 protein.

In yet another embodiment, the present invention encompasses abiological sample derived from a event MIR162 corn plant, tissue, orseed, wherein the sample comprises a nucleotide sequence which is or iscomplementary to a sequence that is unique to event MIR162, and whereinthe sequence is detectable in the sample using a nucleic acidamplification or nucleic acid hybridization method. In one aspect ofthis embodiment, the nucleotide sequence is or is complementary to SEQID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 55, or SEQ ID NO: 59. In another aspect ofthis embodiment, the sample is selected from the group consisting ofcorn flour, corn meal, corn syrup, corn oil, cornstarch, and cerealsmanufactured in whole or in part to contain corn by-products.

In another embodiment, the present invention encompasses an extract of abiological sample derived from a MIR162 corn plant, tissue, or seedcomprising a nucleotide sequence which is or is complementary to asequence that is unique to MIR162. In one aspect of this embodiment, thesequence is detectable in the extract using a nucleic acid amplificationor nucleic acid hybridization method. In another aspect of thisembodiment, the sequence is or is complementary to SEQ ID NO: 1, SEQ IDNO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQID NO: 55, or SEQ ID NO: 59. In yet another aspect of this embodiment,the sample is selected from the group consisting of corn flour, cornmeal, corn syrup, corn oil, cornstarch, and cereals manufactured inwhole or in part to contain corn by-products.

Another embodiment of the present invention encompasses a corn plant, orparts thereof, and seed from a corn plant comprising the genotype of thetransgenic event MIR162, wherein the genotype comprises a nucleotidesequence set forth in SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, orthe complements thereof. One example of corn seed comprising the nucleicacid molecules of the invention was deposited 23 Jan. 2007 and assignedthe ATCC Accession No. PTA-8166. In one aspect of this embodiment, thecorn plant is from the inbred corn lines CG5NA58, CG5NA58A, CG3ND97,CG5NA01, CG5NF22, CG4NU15, CG00685, CG00526, CG00716, NP904, NP911,NP948, NP934, NP982, NP991, NP993, NP2010, NP2013, NP2015, NP2017,NP2029, NP2031, NP2034, NP2045, NP2052, NP2138, NP2151, NP2166, NP2161,NP2171, NP2174, NP2208, NP2213, NP2222, NP2275, NP2276, NP2316, BCTT609,AF031, NPH8431, 894, BUTT201, R327H, 2044BT, and 2070BT. One skilled inthe art will recognize however, that the MIR162 genotype can beintrogressed into any plant variety that can be bred with corn,including wild maize species, and thus the list of inbred lines of thisembodiment are not meant to be limiting.

In another embodiment, the present invention encompasses a corn plantcomprising at least a first and a second DNA sequence linked together toform a contiguous nucleotide sequence, wherein the first DNA sequence iswithin a junction sequence and comprises at least about 11 contiguousnucleotides selected from the group consisting of nucleotides 1079-1098of SEQ ID NO: 49, nucleotides 9381-9400, and the complements thereof,wherein the second DNA sequence is within the heterologous insert DNAsequence set forth in SEQ ID NO: 49, and the complements thereof; andwherein the first and the second DNA sequences are useful as nucleotideprimers or probes for detecting the presence of corn event MIR162nucleic acid sequences in a biological sample. In one aspect of thisembodiment, the nucleotide primers are used in a DNA amplificationmethod to amplify a target DNA sequence from template DNA extracted fromthe corn plant and the corn plant is identifiable from other corn plantsby the production of an amplicon corresponding to a DNA sequencecomprising SEQ ID NO: 45 or SEQ ID NO: 47.

Corn plants of the invention can be further characterized in thatsimultaneously digesting the plant's genomic DNA with the restrictionendonucleases KpnI, EcoRV or NcoI results in an about a 8 kb, a 13 kb or4.6 kb vip3Aa20 hybridizing band, respectively, using a vip3Aa20 probeunder high stringency conditions. Exemplified herein is a vip3Aa20 probecomprising the nucleotide sequence set forth in SEQ ID NO: 13.

Corn plants of the invention can be further characterized in thatdigesting the plant's genomic DNA with the restriction endonucleaseAcc65I or BamHI results in a single pmi hybridizing band using a pmiprobe under high stringency conditions. Exemplified herein is a pmiprobe comprising the nucleotide sequence set forth in SEQ ID NO: 14.

In one embodiment, the present invention provides a corn plant, whereinthe MIR162 genotype confers upon the corn plant insect resistance orability to utilize mannose as a carbon source, or both insect resistanceand the ability to utilize mannose as a carbon source. In one aspect ofthis embodiment, the transgenic genotype conferring insect resistanceupon the corn plant of the invention comprises a vip3Aa20 gene and thetransgenic genotype conferring the ability to utilize mannose as acarbon source upon the maize plant of the invention comprises a pmigene.

In yet another embodiment, the present invention provides a method forproducing a corn plant resistant to lepidopteran pests comprising: (a)sexually crossing a first parent corn plant with a second parent cornplant, wherein said first or second parent corn plant comprises eventMIR162 DNA, thereby producing a plurality of first generation progenyplants; (b) selecting a first generation progeny plant that is resistantto one or more lepidopteran pests; (c) selfing the first generationprogeny plant, thereby producing a plurality of second generationprogeny plants; and (d) selecting from the second generation progenyplants, a plant that is resistant to one or more lepidopteran pests;wherein the second generation progeny plants comprise a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, and SEQ ID NO: 59.

In another embodiment, the present invention provides a method ofproducing hybrid corn seeds comprising: (a) planting seeds of a firstinbred corn line comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 55, and SEQ ID NO: 59, and seeds of a second inbredline having a different genotype; (b) cultivating corn plants resultingfrom said planting until time of flowering; (c) emasculating saidflowers of plants of one of the corn inbred lines; (d) sexually crossingthe two different inbred lines with each other; and (e) harvesting thehybrid seed produced thereby. In one aspect of this embodiment, thefirst inbred corn line provides the female parents. In another aspect ofthis embodiment, the first inbred corn line provides the male parents.The present invention also encompasses the hybrid seed produced by theembodied method and hybrid plants grown from the seed.

One skilled in the art will recognize that the transgenic genotype ofMIR162 can be introgressed by breeding into other corn lines comprisingdifferent transgenic genotypes. For example, a MIR162 corn inbred can becrossed with a corn inbred comprising the transgenic genotype of thelepidopteran resistant Bt11 event (U.S. Pat. Nos. 6,114,608 and6,342,660, herein incorporated by reference). The resulting seed andprogeny plants have the stacked insect resistance traits and thecombined spectrum of activity of Cry1Ab and Vip3Aa20. Another traitstack encompassed by the present invention includes combining the MIR162insect resistance trait and the MIR604 insect resistance trait (USPatent Application publication No. 2005/0216970, published Sep. 29,2005, herein incorporated by reference). The stacked traits in theresulting seed and progeny confer upon the plants an increased spectrumof activity; i.e. the plants are active against both lepidopteran andcoleopteran insect pests.

Therefore, the present invention encompasses a method of protecting atransgenic corn plant from feeding damage by one or more insect pestswherein the method comprises stacking in the same transgenic corn planta Vip3Aa20 insect resistance trait with another insect resistance traitthat is different from Vip3Aa20, whereby the stacked traits protect thecorn plant against feeding damage by one or more insect pests to agreater degree than would be expected due to the insect resistancetraits alone. In one aspect of this embodiment, the Vip3Aa20 insectresistance trait comprised in event MIR162 is stacked with the Cry3A055insect resistance trait comprised in event MIR604 in the same transgeniccorn plant by sexually crossing event MIR162 with event MIR604 or bytransforming the traits together into the same plant.

Examples of other transgenic events which can be crossed with a MIR162inbred include, the glyphosate tolerant GA21 event, the glyphosatetolerant/lepidopteran insect resistant MON802 event, the lepidopteranresistant DBT418 event, the male sterile event MS3, the phosphinothricintolerant event B16, the lepidopteran insect resistant event MON 80100,the phosphinothricin tolerant events T14 and T25, the lepidopteraninsect resistant event 176, and the coleopteran resistant event MON863,all of which are known in the art. It will be further recognized thatother combinations or stacks can be made with the transgenic genotype ofthe invention and thus these examples should not be viewed as limiting.

One skilled in the art will also recognize that transgenic corn seedcomprising the MIR162 genotype can be treated with variousseed-treatment chemicals, including insecticides, to augment orsyngergize the insecticidal activity of the Vip3Aa20 protein.

The subject invention discloses herein a specific site on chromosome 5in the maize genome that is excellent for insertion of heterologousnucleic acids. Also disclosed is a 5′ molecular marker (opie2;nucleotides 1680-3338 of SEQ ID NO: 106) and a 3′ molecular marker (gag;nucleotides 43,275-45,086 of SEQ ID NO: 106) useful in identifying thelocation of a targeting site on chromosome 5. Thus, the subjectinvention provides methods to introduce heterologous nucleic acids ofinterest into this pre-established target site or in the vicinity ofthis target site. The subject invention also encompasses a corn seedand/or a corn plant comprising any heterologous nucleotide sequenceinserted at the disclosed target site or in the general vicinity of suchsite. One option to accomplish such targeted integration is tosubstitute a different insert in place of the vip3Aa20 expressioncassette exemplified herein. In this general regard, targeted homologousrecombination, for example without limitation, can be used according tothe subject invention. “Homologous recombination” refers to a reactionbetween any pair of nucleotide sequences having corresponding sitescontaining a similar nucleotide sequence (i.e., homologous sequences)through which the two molecules can interact (recombine) to form a new,recombinant DNA sequence. The sites of similar nucleotide sequence areeach referred to herein as a “homology sequence”. Generally, thefrequency of homologous recombination increases as the length of thehomology sequence increases. Thus, while homologous recombination canoccur between two nucleotide sequences that are less than identical, therecombination frequency (or efficiency) declines as the divergencebetween the two sequences increases. Recombination may be accomplishedusing one homology sequence on each of the donor and target molecules,thereby generating a “single-crossover” recombination product.Alternatively, two homology sequences may be placed on each of thetarget and donor nucleotide sequences. Recombination between twohomology sequences on the donor with two homology sequences on thetarget generates a “double-crossover” recombination product. If thehomology sequences on the donor molecule flank a sequence that is to bemanipulated (e.g., a sequence of interest), the double-crossoverrecombination with the target molecule will result in a recombinationproduct wherein the sequence of interest replaces a DNA sequence thatwas originally between the homology sequences on the target molecule.The exchange of DNA sequence between the target and donor through adouble-crossover recombination event is termed “sequence replacement.”This type of technology is the subject of, for example, US patentApplication Publication No. 2006/0253918, herein incorporated byreference. With the disclosed target site now being identified and withthe sequences surrounding the identified target site, the skilled personwill recognize that other methods for targeted integration ofheterologous nucleic acids may be used. Such methods, for examplewithout limitation, are disclosed in US Patent Application PublicationNo. 2007/0039074 and US Patent Application Publication No. 2006/0130179.

In one embodiment, the present invention encompasses a maize chromosomaltarget site located on chromosome 5 between a opie2 molecular marker setforth as nucleotides 1680-3338 of SEQ ID NO: 106 and a gag molecularmarker set forth as nucleotides 43,275-45,086 of SEQ ID NO: 106, whereinthe target site comprises a heterologous nucleic acid. In anotherembodiment, the maize chromosomal target site is located on chromosome 5between nucleotides 25,454 and 25,513 of SEQ ID NO: 106. In yet anotherembodiment, the chromosomal target site is flanked 5′ by nucleotides5,454 to 25,454 of SEQ ID NO: 106 and flanked 3′ by nucleotides 25,513to 45,513 of SEQ ID NO: 106.

In one embodiment, the present invention encompasses a method of makinga transgenic maize plant comprising inserting a heterologous nucleicacid at a position on chromosome 5 located between a opie2 molecularmarker set forth as nucleotides 1680-3338 of SEQ ID NO: 106 and a gagmolecular marker set forth as nucleotides 43,275-45,086 of SEQ ID NO:106. In another embodiment, the heterologous nucleic acid is inserted onchromosome 5 between nucleotides 25,454 and 25,513 of SEQ ID NO: 106. Instill another embodiment, the inserted heterologous nucleic acid isflanked 5′ by nucleotides 5,454 to 25,454 of SEQ ID NO: 106 and flanked3′ by nucleotides 25,513 to 45,513 of SEQ ID NO: 106

The transgenic genotype of the present invention can be introgressed inany corn inbred or hybrid using art recognized breeding techniques. Thegoal of plant breeding is to combine in a single variety or hybridvarious desirable traits. For field crops, these traits may includeresistance to insects and diseases, tolerance to herbicides, toleranceto heat and drought, reducing the time to crop maturity, greater yield,and better agronomic quality. With mechanical harvesting of many crops,uniformity of plant characteristics such as germination and standestablishment, growth rate, maturity, and plant and ear height, isimportant.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Corn (Zea mays L.), can be bred by both self-pollination andcross-pollination techniques. Corn has separate male and female flowerson the same plant, located on the tassel and the ear, respectively.Natural pollination occurs in corn when wind blows pollen from thetassels to the silks that protrude from the tops of the ears.

A reliable method of controlling male fertility in plants offers theopportunity for improved plant breeding. This is especially true fordevelopment of corn hybrids, which relies upon some sort of malesterility system. There are several options for controlling malefertility available to breeders, such as: manual or mechanicalemasculation (or detasseling), cytoplasmic male sterility, genetic malesterility, gametocides and the like.

Hybrid corn seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using one of many methods of conferring genetic malesterility in the art, each with its own benefits and drawbacks. Thesemethods use a variety of approaches such as delivering into the plant agene encoding a cytotoxic substance associated with a male tissuespecific promoter or an antisense system in which a gene critical tofertility is identified and an antisense to that gene is inserted in theplant (see: Fabinjanski, et al. EPO 89/3010153.8 publication no. 329,308and PCT application PCT/CA90/00037 published as WO 90/08828).

The use of male sterile inbreds is but one factor in the production ofcorn hybrids. Plant breeding techniques known in the art and used in acorn plant breeding program include, but are not limited to, recurrentselection, backcrossing, pedigree breeding, restriction lengthpolymorphism enhanced selection, genetic marker enhanced selection andtransformation. The development of corn hybrids in a corn plant breedingprogram requires, in general, the development of homozygous inbredlines, the crossing of these lines, and the evaluation of the crosses.Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Corn plant breedingprograms combine the genetic backgrounds from two or more inbred linesor various other germplasm sources into breeding pools from which newinbred lines are developed by selfing and selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which of thosehave commercial potential. Plant breeding and hybrid development, aspracticed in a corn plant-breeding program, are expensive andtime-consuming processes.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding five or more generations ofselfing and selection is practiced: F₁→F₂; F₂→F₃; F₃→F₄; F₄→F.₅; etc.

Recurrent selection breeding, backcrossing for example, can be used toimprove an inbred line and a hybrid that is made using those inbreds.Backcrossing can be used to transfer a specific desirable trait from oneinbred or source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred(recurrent parent) to a donor inbred (non-recurrent parent), thatcarries the appropriate gene(s) for the trait in question. The progenyof this cross is then mated back to the superior recurrent parentfollowed by selection in the resultant progeny for the desired trait tobe transferred from the non-recurrent parent. After five or morebackcross generations with selection for the desired trait, the progenywill be homozygous for loci controlling the characteristic beingtransferred, but will be like the superior parent for essentially allother genes. The last backcross generation is then selfed to give purebreeding progeny for the gene(s) being transferred. A hybrid developedfrom inbreds containing the transferred gene(s) is essentially the sameas a hybrid developed from the same inbreds without the transferredgene(s).

Elite inbred lines, that is, pure breeding, homozygous inbred lines, canalso be used as starting materials for breeding or source populationsfrom which to develop other inbred lines. These inbred lines derivedfrom elite inbred lines can be developed using the pedigree breeding andrecurrent selection breeding methods described earlier. As an example,when backcross breeding is used to create these derived lines in a cornplant-breeding program, elite inbreds can be used as a parental line orstarting material or source population and can serve as either the donoror recurrent parent.

A single cross corn hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F₁. In thedevelopment of commercial hybrids in a corn plant-breeding program, onlythe F₁ hybrid plants are sought. Preferred F₁ hybrids are more vigorousthan their inbred parents. This hybrid vigor, or heterosis, can bemanifested in many polygenic traits, including increased vegetativegrowth and increased yield.

The development of a corn hybrid in a corn plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, although different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrid progeny (F₁). Duringthe inbreeding process in corn, the vigor of the lines decreases. Vigoris restored when two different inbred lines are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred lines is that the hybrid between a definedpair of inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

A single cross hybrid is produced when two inbred lines are crossed toproduce the F₁ progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F₁ hybridsare crossed again (A×B)×(C×D). A three-way cross hybrid is produced fromthree inbred lines where two of the inbred lines are crossed (A×B) andthen the resulting F₁ hybrid is crossed with the third inbred (A×B)×C.Much of the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrids is not used forplanting stock.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female parent. Incomplete removal or inactivation of thepollen provides the potential for self-pollination. This inadvertentlyself-pollinated seed may be unintentionally harvested and packaged withhybrid seed.

Once the seed is planted, it is possible to identify and select theseself-pollinated plants. These self-pollinated plants will be geneticallyequivalent to the female inbred line used to produce the hybrid.

Typically these self-pollinated plants can be identified and selecteddue to their decreased vigor. Female selfs are identified by their lessvigorous appearance for vegetative and/or reproductive characteristics,including shorter plant height, small ear size, ear and kernel shape,cob color, or other characteristics.

Identification of these self-pollinated lines can also be accomplishedthrough molecular marker analyses. See, “The Identification of FemaleSelfs in Hybrid Maize: A Comparison Using Electrophoresis andMorphology”, Smith, J. S. C. and Wych, R. D., Seed Science andTechnology 14, pp. 1-8 (1995), the disclosure of which is expresslyincorporated herein by reference. Through these technologies, thehomozygosity of the self-pollinated line can be verified by analyzingallelic composition at various loci along the genome. Those methodsallow for rapid identification of the invention disclosed herein. Seealso, “Identification of Atypical Plants in Hybrid Maize Seed byPostcontrol and Electrophoresis” Sarca, V. et al., Probleme de GeneticaTeoritica si Aplicata Vol. 20 (1) p. 29-42.

As is readily apparent to one skilled in the art, the foregoing are onlysome of the various ways by which the inbred of the present inventioncan be obtained by those looking to introgress the transgenic genotypeof the invention into other corn lines. Other means are available, andthe above examples are illustrative only.

The following examples are intended solely to illustrate one or morepreferred embodiments of the invention and are not to be construed aslimiting the scope of the invention.

EXAMPLES Example 1. Transformation and Selection of the MIR162 Event

The MIR604 event was produced by Agrobacterium-mediated transformationof a proprietary corn (Zea mays) line Immature embryos were transformedessentially as described in Negrotto et al. (Plant Cell Reports 19:798-803, 2000), incorporated herein by reference, using a DNA fragmentfrom plasmid pNOV1300 (SEQ ID NO: 3). pNOV1300 contains a nucleotidesequence comprising tandem expression cassettes. The first expressioncassette comprises a ZmUbiInt promoter region from a Zea mayspolyubiquitin gene, which contains the first intron (GenBank® Accessionnumber S94464) operably linked to a vip3Aa19 coding sequence furtheroperably linked to PEPC Intron #9 from the phosphoenolpyruvatecarboxylase gene (GenBank® Accession Number X15239) from Zea mays(Matsuoka and Minami, 1989. European J. Of Biochem. 181:593-598) and a35S terminator sequence from the 35S RNA from the cauliflower mosaicvirus genome (Similar to GenBank® Accession Number AF140604). Itsfunction is to provide a polyadenylation sequence (Franck et al., 1980.Cell 21:285-294). The vip3Aa19 gene in pNOV1300 comprises a syntheticmaize-optimized vip3Aa coding sequence (Estruch, et al., 1999.) whichwas synthesized to accommodate the preferred codon usage for maize(Murray et al., 1989). The synthetic vip3Aa19 coding sequence used inplant transformations encodes the identical amino acid sequence as thenative vip3Aa1 coding sequence found in the soil bacterium Bacillusthuringiensis strain AB88 (U.S. Pat. No. 5,877,012), with the exceptionof a single amino acid difference at position 284; the native vip3Aa1coding sequence encodes lysine, whereas the synthetic vip3Aa19 codingsequence encodes glutamine at this position. The vip3Aa19 codingsequence encodes an insect control protein, Vip3Aa19 that providesresistance to lepidopteran insects. The second expression cassette iscomprised of a ZmUbiInt promoter operably linked to a pmi codingsequence (also known as E. coli manA) encoding phosphomannose isomerase(GenBank® Accession number M15380), which catalyzes the isomerization ofmannose-6-phosphate to fructose-6-phosphate (Negrotto et al., 2000). Thepmi coding sequence is further operably linked to a nopaline synthase 3′end transcription termination and polyadenylation sequence.

Immature embryos were excised from 8-12 day old ears and rinsed withfresh medium in preparation for transformation. Embryos were mixed withthe suspension of Agrobacterium cells harboring the transformationvector pNOV1300, vortexed for 30 seconds, and allowed to incubate for anadditional 5 minutes. Excess solution containing Agrobacterium wasaspirated and embryos were then moved to plates containing anon-selective culture medium. Embryos were co-cultured with theremaining Agrobacterium at 22° C. for 2-3 days in the dark. Embryos weretransferred to culture medium supplemented with ticarcillin (100 mg/ml)and silver nitrate (1.6 mg/l) and incubated in the dark for 10 days.Embryos producing embryogenic callus were transferred to cell culturemedium containing mannose.

Regenerated plantlets were tested by TAQMAN® PCR analysis (see Example2) for the presence of both the pmi and vip3Aa19 genes, as well as forthe absence of the antibiotic resistance spectinomycin (spec) gene. Itwas later discovered (See Example 4 below) that during thetransformation process two mutations were introduced into the vip3Aa19coding sequence, one of which resulted in an amino acid change in theVip3Aa19 protein. Therefore, this new vip3Aa coding sequence, which isunique to event MIR162, was designated vip3Aa20. The vip3Aa20 codingsequence encodes isoleucine at position 129 in place of the methionineresidue encoded by the vip3Aa19 gene.

Plants positive for both transgenes, and negative for the spec gene,were transferred to the greenhouse for further propagation. Positiveevents were identified and screened using insect bioassays against fallarmyworm. Insecticidal events were characterized for copy number byTAQMAN analysis. MIR162 was chosen for further analysis based on havinga single copy of the transgenes, good protein expression as identifiedby ELISA, and good insecticidal activity against fall armyworm.

The breeding pedigree of the MIR162 event was as follows: T₀ MIR162plant (x NPH8431)→→NPH8431 (MIR162) F₁ (x NP2161)→NP2161(MIR162) F₁ (xNP2161)→NP2161 (MIR162) BC1F₁ (x B9620)→F₁ (x B9620)→BC1F₁ (xB9620)→BC2F₁ (x B9620)→BC3F₁ (x B9620)→BC4F₁ (x B9620). Plant materialfrom the BC4 generation was used for the Southern analysis, copy numberdetermination and sequencing of the insert DNA. Negative controls forthe experiments consisted of 10 negative segregant plants from the BC4generation.

Example 2. MIR162 Detection by TAQMAN PCR

TAQMAN analysis was essentially carried out as described in Ingham etal. (Biotechniques, 31:132-140, 2001) herein incorporated by reference.Briefly, genomic DNA was isolated from leaves of transgenic andnon-transgenic corn plants using the Puregene® Genomic DNA Extractionkit (Gentra Systems, Minneapolis, Minn.) essentially according to themanufacturer's instruction, except all steps were conducted in 1.2 ml96-well plates. The dried DNA pellet was resuspended in TE buffer (10 MmTris-HCl, pH 8.0, 1 mM EDTA).

TAQMAN PCR reactions were carried out in 96-well plates. For theendogenous corn gene control, primers and probes were designed specificto the Zea mays alcohol dehydrogenase (adhI) coding sequence (Genbankaccession no. AF044295). It will be recognized by the skilled personthat other corn genes can be used as endogenous controls. Reactions weremultiplexed to simultaneously amplify vip3Aa and adhI or pmi and adhI.For each sample, a master mixture was generated by combining 20 μLextracted genomic DNA with 35 μL 2× TAQMAN Universal PCR Master Mix(Applied Biosystems) supplemented with primers to a final concentrationof 900 nM each, probes to a final concentration of 100 nM each, andwater to a 70 μL final volume. This mixture was distributed into threereplicates of 20 μL each in 96-well amplification plates and sealed withoptically clear heat seal film (Marsh Bio Products). PCR was run in theABI Prism 7700 instrument using the following amplification parameters:2 min at 50° C. and 10 min at 95° C., followed by 35 cycles of 15 s at95° C. and 1 min at 60° C.

Results of the TAQMAN analysis demonstrated that event MIR162 had onecopy of the vip3Aa20 gene and one copy of the pmi gene.

Primers and probes that were used in the TAQMAN PCR reactions are shownin Table 1.

TABLE 1 Primers used in TAQMAN Assay. Primer Name Primer SequenceSequence No: Vip3Aa-forward 5′CACCTTCAGCAACCCGAACTA3′ SEQ ID NO: 4Vip3Aa-reverse 5′GCTTAGCCTCCACGATCATCTT3′ SEQ ID NO: 5 Vip3Aa-probe5′GTCCTCGTCGCTGCCCTTCACCT3′ SEQ ID NO: 6 (5′ label = FAM, 3′ label= TAMRA) PMI-forward 5′CCGGGTGAATCAGCGTTT3′ SEQ ID NO: 7 PMI-reverse5′GCCGTGGCCTTTGACAGT3′ SEQ ID NO: 8 PMI-probe 5′TGCCGCCAACGAATCACCGG3′SEQ ID NO: 9 (5′ label = FAM, 3′label = TAMRA) ZmADH-267forward5′GAACGTGTGTTGGGTTTGCAT3′ SEQ ID NO: 10 ZmADH-337 reverse5′TCCAGCAATCCTTGCACCTT3′ SEQ ID NO: 11 ZmADH-316 probe5′TGCAGCCTAACCATGCGCAGGGTA3′ SEQ ID NO: 12 (5′label = TET, 3′ label= TAMRA)

Example 3. MIR162 Detection by Southern Blot

Genomic DNA used for southern analysis was isolated from pooled leaftissue of 10 plants representing the BC4 generation of MIR162 usingessentially the method of Thomas et al. (Theor. Appl. Genet. 86:173-180,1993), incorporated herein by reference. All plants used for DNAisolation were individually analyzed using TAQMAN PCR (as described inExample 2) to confirm the presence of a single copy of the vip3Aa20 geneand the pmi gene. For the negative segregant controls, DNA was isolatedfrom pooled leaf tissue of negative segregants from the BC4 generation.These negative segregant plants were individually analyzed using TAQMANPCR to confirm the absence of the vip3Aa20 and pmi genes, but were, asexpected, positive for the endogenous maize adhI gene.

Southern analysis was carried out using conventional molecular biologytechniques. (See Chomczynski, P. 1992. Analytical Biochemistry201:34-139) Genomic DNA (7.5 μg) was digested with restriction enzymesthat digest within the event MIR162 insert, but not within the codingsequence that corresponds to the specific probe used in the experiment.This approach allowed for determination of the number of copies of eachgene, corresponding to the specific probe used for each Southernanalysis, which was incorporated into event MIR162.

Another series of restriction digests was performed in which the insertwas digested with restriction enzymes that would release a fragment ofknown size from the insert. This approach provided additional evidencefor the presence of a single copy of each coding sequence present inMIR162 and allowed for the detection of partial copies of the insertthat may be closely linked to the MIR162 insert. Following agarose gelelectrophoresis and alkaline transfer to a ZetaProbe® GT membrane(Bio-Rad, Cat. No. 162-0195), hybridizations were carried out usingfull-length PCR generated element probes. The probes were labeled with³²P via random priming using the MegaPrime™ system (AmershamBiosciences, Cat. No. RPN1607). Hybridization was carried out at 65° C.,followed by multiple washes in 2×SSC, 0.1% SDS and then 0.1×SSC and 0.1%SDS. The membranes were then subjected to autoradiography.

Included in each Southern analysis were three control samples: (1) DNAfrom a negative (non-transformed) segregant used to identify anyendogenous Zea mays sequences that may cross-hybridize with theelement-specific probe; (2) DNA from a negative segregant into which isintroduced an amount of digested pNOV1300 that is equal to one copynumber based on plasmid size was introduced, to demonstrate thesensitivity of the experiment in detecting a single gene copy within theZea mays genome; and (3) Digested pNOV1300 plasmid equal to one copynumber based on plasmid size, to act as a positive control forhybridization as well as to demonstrate the sensitivity of theexperiment.

The results of Southern analyses demonstrated that the MIR162 insertcontains a single copy of the vip3Aa20 gene and pmi gene and contains nopNOV1300 backbone sequences. A vip3Aa19 probe (SEQ ID NO: 13) was usedfor the vip3Aa20 Southern analysis. The nucleotide sequences of vip3Aa19and vip3Aa20 differ by two nucleotides and are 99.9% identical.Therefore, the vip3Aa19 probe hybridized to the vip3Aa20 sequencepresent in MIR162 under stringent conditions. Using the vip3Aa19 probe,a KpnI and an EcoRV digest resulted in single hybridization bandsapproximately 8 kb and 13 kb in size, respectively. In addition, an NcoIdouble digest resulted in a single hybridization band consistent withthe expected size of 4.6 kb. Using the pmi probe (SEQ ID NO: 14), aAcc65I and a BamHI digest resulted in single hybridization bands ofapproximately 4 kb and 6 kb in size, respectively. In addition, anXmaI+HindIII double digest resulted in a single hybridization bandconsistent with the expected size of 8.1 kb. The 8.1 kb XmaI+HindIIIpNOV1300 band (positive control) also hybridized with the vip3Aa19 andpmi probes as expected. Some cross-hybridization in the plasmid-onlylanes with the DNA ladder probe was detected. Typically commerciallyavailable DNA ladders may contain some vector sequences that cancross-hybridize with the plasmid control sequences as observed in theseexperiments, but, this does not impact the findings of this study.Finally, a pNOV1300 backbone probe did not hybridize demonstrating theabsence of incorporation of any pNOV1300 vector backbone sequences intoMIR162 during the transformation process.

Example 4. Heterologous DNA Insert Sequencing

The nucleotide sequence of the vip3Aa and pmi coding sequences in theheterologous DNA molecule inserted in MIR162 was determined todemonstrate overall integrity of the insert, contiguousness of thefunctional elements and to detect any individual basepair changes. Thecoding sequences were amplified from DNA derived from the BC4generation. PCR amplification was carried out using either Expand HighFidelity PCR system (Roche, Cat. No. 1732650) or PfuUltra™ HotstartHigh-Fidelity DNA polymerase (Stratagene, Cat. No. 600390). Each PCRproduct was individually cloned into either pCR®-XL-TOPO vector(Invitrogen, Cat. No. K4700-20) or pCR®-BluntII-TOPO vector (Invitrogen,Cat. No. K2800-20) and three separate clones for each PCR product wereidentified and sequenced. Sequencing was carried out using the ABI3730XLanalyzer using ABI BigDye® 1.1 or Big Dye 3.1 dGTP (for GC-richtemplates) chemistry. The sequence analysis was done using the Phred,Phrap, and Consed package from the University of Washington and wascarried out to an error rate of less than 1 in 10,000 bases (Ewing &Green, 1998. Genome Research 8:186-194). The final consensus sequencefor each gene was determined by combining the sequence data from thethree individual clones to generate one consensus sequence for eachgene. Sequence alignment was performed using the ClustalW program withthe following parameters: scoring matrix blosum55, gap opening penalty15, gap extension penalty 6.66 (Thompson et al, 1994. Nucleic AcidsResearch 22:4673-4680).

The full vip3Aa20 coding sequence was PCR amplified using primersMOV3Aa-01-5′: 5′ATGAACAAGAACAACACCAA3′ (SEQ ID NO: 15) and MOV3Aa-01-3′:5′CTACTTGATGCTCACGTCGTAG3′ (SEQ ID NO: 16) and PfuUltra Hotstart enzymegenerating a 2370 bp product. The PCR amplicon was sequenced using theprimers shown in Table 2.

TABLE 2 Primer Name quence (5′→3′) Sequence No. b03503bACGAGCAGAACCAGGTGC SEQ ID NO: 17 b03503c GGTGAAGAAGGACGGCAG SEQ ID NO:18 b03503d ACCTGTCGCAAGCTGCTGGG SEQ ID NO: 19 b03503e TGGACAAGCTGCTGTGTCSEQ ID NO: 20 b03503f TGCAGGCCGACGAGAACAG SEQ ID NO: 21 b03503gTGATCCAGTACACCGTGAA SEQ ID NO: 22 b03503h ACCCTGACCCTGTACCAG SEQ ID NO:23 b03504b GTGTTGCCGCTGATGTTG SEQ ID NO: 24 b03504c CGTACTCGGTCTTCGGCTSEQ ID NO: 25 b03504d CTGCAGGCCAAAGCCGTT SEQ ID NO: 26 b03504eTCGCCGTAGATCACCTCG SEQ ID NO: 27 b03504f GCTTGCGACAGGTGGTCA SEQ ID NO:28 b03504g TTGCTGCTGGTCTCGGTGG SEQ ID NO: 29 b03504h CGTTGGCGATCTTAAGGATSEQ ID NO: 30 b00203c GCAAGCCATCGATTCAC SEQ ID NO: 31 b00203dGCAACACCCTGACCCTG SEQ ID NO: 32 b00203e TCTACGACGTGAGCATCAAG SEQ ID NO:33 b00203f GTAGAAGTGCACGATCGGG SEQ ID NO: 34 b00203g CGGTGCTGGTCCAGTTGSEQ ID NO: 35

Two other PCR reactions overlapped the full vip3Aa20 coding sequence.The 5′ end of vip3Aa20 was covered with a PCR amplification usingprimers 162INSERT-F2: 5′ACACCAATGATGCAAATAGGC3′ (SEQ ID NO: 36) andVIP_R4 5′GAAGGTGTTCAGGTAGAACTCGAAG3′ (SEQ ID NO: 37) and Expand HighFidelity enzyme. The second reaction covered the 3′ end of vip3Aa20; theproduct was amplified with primers VIP-F3: 5′GGTGCTGTTCGAGAAGAGGT3′ (SEQID NO: 42) and PMI_REV1: 5′CGATTTATCACTCTCAATCACAT3′ (SEQ ID NO: 43) andExpand High Fidelity enzyme. The amplicons generated by these reactionscomprised a 2946 bp nucleotide sequence (SEQ ID NO: 38) and a 2577 bpnucleotide sequence (SEQ ID NO: 44), respectively.

The consensus sequence data revealed two nucleotide changes in thevip3Aa coding sequence in MIR162 (designated vip3Aa20; SEQ ID NO: 1)compared to the vip3Aa coding sequence in pNOV1300 (designatedvip3Aa19), which was used to transform MIR162. The first nucleotidechange, a G to T mutation, occurred at position 387 of the vip3Aa19coding sequence (SEQ ID NO: 3). This mutation resulted in the methionineat position 129 of Vip3Aa19 being changed to isoleucine in Vip3Aa20(M129I), SEQ ID NO: 2. The second nucleotide change occurred at position1683 of the coding sequence, a G to C mutation, but did not result in anamino acid change. Therefore, the vip3Aa20 coding sequence (SEQ IDNO: 1) and the Vip3Aa20 protein (SEQ ID NO: 2) are unique to the MIR162event and can be used to identify any plant comprising the MIR162transgenic genotype. The pmi coding sequence MIR162 was identical tothat in the transformation plasmid pNOV 1300. An alignment of theVip3Aa20 and Vip3Aa19 insecticidal proteins is shown in Table 3.

TABLE 3 Comparison of Vip3Aa20 and Vip3Aa19 amino acid sequences. NameSequence Alignment Vip3Aa20  (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE Vip3Aa19  (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE Vip3Aa20 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL Vip3Aa19 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL Vip3Aa20Vip3Aa19

Vip3Aa20 (151) DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDGVip3Aa19 (151) DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDGVip3Aa20 (201) SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKVip3Aa19 (201) SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKVip3Aa20 (251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLADVip3Aa19 (251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLADVip3Aa20 (301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEVip3Aa19 (301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEVip3Aa20 (351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLLVip3Aa19 (351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLLVip3Aa20 (401) CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIVip3Aa19 (401) CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIVip3Aa20 (451) DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSVip3Aa19 (451) DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSVip3Aa20 (501) RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLEVip3Aa19 (501) RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLEVip3Aa20 (551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYTVip3Aa19 (551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYTVip3Aa20 (601) VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILKVip3Aa19 (601) VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILKVip3Aa20 (651) SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLYVip3Aa19 (651) SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLYVip3Aa20 (701) QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAKVip3Aa19 (701) QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAKVip3Aa20 (751) DVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK Vip3Aa19(751) DVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK The shaded box indicatesthe amino acid change.

Example 5. Analysis of Flanking DNA Sequence

A number of methods are known to those of skill in the art to amplifyunknown DNA sequences adjacent to a core region of known sequence. Thosemethods include, but are not limited to, inverse PCR (iPCR) [Ochman et.al., Genetics 120:621-623 (1988); Triglia et. al., Nucleic Acids Res.16:8186 (1988)], panhandle PCR [Jones and Winistorfer, Nucleic AcidsRes. 20:595-600 (1992); Jones and Winistorfer, Biotechniques 23:132-138(1997)], cassette ligation-anchored PCR [Mueller and Wold, Science246:780-786 (1989)], vectorette-PCR [Riley et. al., Nucleic Acids Res.18:2887-2890 (1990)], novel-Alu-PCR [Puskas et. al., Nucleic Acids Res.22:3251-3252 (1994)] and Thermal Asymmetric Interlaced PCR (TAIL-PCR)[Liu and Whittier, Genomics 25:673-681 (1995)].

One method used to amplify corn genome DNA sequence flanking theheterologous DNA inserted into event MIR162 was vectorette PCRessentially as described by Riley et al., Nucleic Acids Res.18:2887-2890 (1990), incorporated herein by reference.

The 5′ flanking sequence and junction sequence was confirmed usingstandard PCR procedures. The following primer pairs, or complementsthereof, were used to confirm the sequence: 162INSERT-F2:5′ACACCAATGATGCAAATAGGC3′ (SEQ ID NO: 36)/VIP_R4:5′GAAGGTGTTCAGGTAGAACTCGAAG3′ (SEQ ID NO: 37) and CJB 179:5′ATGCAAATAGGCTGGGAATAGTC3′ (SEQ ID NO: 39)/CJB 1345′GTACCAGCTTGCTGAGTGGCT3′ (SEQ ID NO: 40). The resulting amplicon hasthe sequence shown is SEQ ID NO: 41 and comprises the 5′ junctionsequence of SEQ ID NO: 45. It will be recognized that other primersequences can be used to confirm the flanking and junction sequences.Using this method, the MIR162 insert was found to be flanked 5′ bynucleotides 1040-1088 of the corn genomic sequence shown in SEQ ID NO:46.

A larger region of the 5′ flanking sequence from event MIR162 wasgenerated using the Seegene DNA Walking SpeedUp™ Premix kit followingthe manufacturer's instructions.

A first PCR reaction was performed independently in four individualtubes using primer FE1002: 5′CGTGACTCCCTTAATTCTCCGCT3′ (SEQ ID NO: 50)with one of the DW-ACP 1, 2, 3, or 4 primers supplied by themanufacturer. The following reagents were mixed in a PCR tube on ice:100 μg MIR162 genomic DNA, 4 μl 2.5 μM DW-ACP (one each with DW-ACP 1,2, 3, or 4), 4 μl 2.5 μM FE1002, 19 μl distilled water, and 25 μl 2×SeeAmp™ ACP™ Master Mix II. The tubes were placed in a preheated (94°C.) thermal cycler. PCR was completed using the following program: onecycle at 94° C. for five minutes, 42° C. for one minute, and 72° C. fortwo minutes, 30 cycles of 94° C. for 40 seconds, 55° C. for 40 seconds,and 72° C. for 90 seconds, and one cycle at 72° C. for seven minutes.The PCR products were purified using Exonuclease I and Shrimp AlkalinePhosphatase.

A second PCR reaction was performed independently in four individualtubes using primer FE1003: 5′GATCAGATTGTCGTTTCCCGCCTT3′ (SEQ ID NO: 51)with the DW-ACPN primer supplied by the manufacturer of the kit. Thefollowing reagents were mixed in a PCR tube on ice: 3 μl purified PCRproduct, 1 μl 10 μM DW-ACPN, 1 μl 10 μM FE1003, 5 μl distilled water,and 10 μl 2× SeeAmp™ ACP™ Master Mix II. The tubes were placed in apreheated (94° C.) thermal cycler. PCR was completed using the followingprogram: one cycle at 94° C. for five minutes, 35 cycles of 94° C. for40 seconds, 60° C. for 40 seconds, and 72° C. for 90 seconds, and onecycle at 72° C. for seven minutes.

A third PCR reaction was performed independently in four individualtubes using primer FE1004: 5′GATTGTCGTTTCCCGCCTTCAGTT3′ (SEQ ID NO: 52)with the Universal primer supplied by the manufacturer. The followingreagents were mixed in a PCR tube on ice: 2 μl purified PCR product, 1μl 10 μM Universal primer, 1 μl 10 μM FE1004, 6 μl distilled water, and10 μl 2× SeeAmp™ ACP™ Master Mix II. The tubes were placed in apreheated (94° C.) thermal cycler. PCR was completed using the followingprogram: one cycle at 94° C. for five minutes, 35 cycles of 94° C. for40 seconds, 60° C. for 40 seconds, and 72° C. for 90 seconds, and onecycle at 72° C. for seven minutes.

Ten μl of the PCR products were run on a 1% agarose gel containingethidium bromide. The appropriate band was extracted from the agarosegel and purified using a Qiagen Qiaquick Gel Extraction Kit according tothe manufacturer's instructions. The extracted DNA was cloned into anInvitrogen TOPO-XL cloning vector according to the manufacturer'sinstructions. This clone was transformed into E. coli, and the plasmidDNA was extracted from the cells after overnight growth with a QiagenMiniprep kit according to the manufacturer's instructions. This plasmidwas used for end run sequencing.

A new primer was designed within the new, previously unknown sequence tobe used with a primer in the heterologous DNA insert to amplify the full1 kb of flanking sequence out of the genomic DNA. Flanking sequenceprimer 162DWConf3: 5′CCTGTGTTGTTGGAACAGACTTCTGTC3′ (SEQ ID NO: 53) andinsert DNA primer FE0900: 5′GGCTCCTTCAACGTTGCGGTTCTGTC3′ (SEQ ID NO: 54)were used to amplify a nucleic acid molecule comprising the 5′ flankingsequence for confirmation. The sequence of the resulting amplicon is setforth in SEQ ID NO: 55. This 5′ amplicon comprises the 5′ junctionsequence set forth in SEQ ID NO: 45. Ten μl of the PCR product(amplicon) was run on a 1% agarose gel containing ethidium bromide. Theappropriate band was extracted from the agarose gel and purified using aQiagen Qiaquick Gel Extraction Kit according to the manufacturer'sinstructions. The extracted DNA was cloned into an Invitrogen TOPO-XLcloning vector according to the manufacturer's instructions. This clonewas transformed into E. coli. Plasmid DNA was extracted from the cellsafter overnight growth in media with a Qiagen Miniprep kit according tothe manufacturer's instructions. Three plasmids were completelysequenced using the primers shown in Table 4. The plasmid sequences werealigned to generate the complete confirmed 5′ flanking sequence. Usingthis method, approximately 1 kb of the 5′ flanking sequence (SEQ ID NO:46) was determined

TABLE 4 Primer sequences. Primer Name Sequence (5′→3′) Sequence No.b00201h TTCACGGGAGACTTTATCTG SEQ ID NO: 60 b00605a CCGATTCATTAATGCAG SEQID NO: 61 b00701b ACGTAAAACGGCTTGTC SEQ ID NO: 62 b00702bGTTTAAACTGAAGGCGG SEQ ID NO: 63 b00704h AATAATATCACTCTGTACATCC SEQ IDNO: 64 b01106f GTTGTAAAACGACGG SEQ ID NO: 65 b01709f TAGGCACCCCAGGCTTTASEQ ID NO: 66 b03504a AATTGAATTTAGCGGCCG SEQ ID NO: 67 b05102fGGTCCCTACAACATAAATAG SEQ ID NO: 68 b05102g TTCGTCCCTACTATCAACGC SEQ IDNO: 69 b05102h CTTTAGGCATCAGCGGGT SEQ ID NO: 70 b05103aAGCATCTGCGTAAGCACA SEQ ID NO: 71 b05103b CTGATGACACCAATGATGC SEQ ID NO:72 b05103c GATCAGATTGTCGTTTCCC SEQ ID NO: 73 b05103d GCATCATTGGTGTCATCAGSEQ ID NO: 74 b05103e TGTGCTTACGCAGATGCT SEQ ID NO: 75 b05103fACCCGCTGATGCCTAAAG SEQ ID NO: 76 b05103g GCGTTGATAGTAGGGACGAA SEQ ID NO:77 b05103h CTATTTATGTTGTAGGGACC SEQ ID NO: 78 b05210a CTAGACTGGAAAGCGGAGSEQ ID NO: 79 b05210b CCACTTTCATCCCTAGTTG SEQ ID NO: 80

The 3′ flanking sequence from event MIR162 was generated using theClonetech GenomeWalker™ Universal kit (Clonetech Laboratories, Inc.)following the manufacturer's instructions.

First, pools of uncloned, adaptor-ligated genomic DNA fragments, knownas GenomeWalker “libraries” were constructed. Each library wasconstructed by digesting the MIR162 genomic DNA with a restrictionenzyme (DraI, EcoRV, PvuII, StuI, and XmnI) as follows: For example, 25μl MIR162 genomic DNA (0.1 μg/μl), 8 μl restriction enzyme (10units/μl), 10 μl restriction enzyme buffer (10×), and 57 μl distilledH2O were mixed in a tube and incubated at 37° C. overnight.

DNA was then purified by using several rounds of phenol/chloroformextraction. Finally, the DNA was precipitated and washed with ethanol,dried and dissolved into 20 μl of TE buffer.

To ligate the GenomeWalker Adapter ends to the MIR162 genomic DNA, 4 μlof the digested, purified genomic DNA was mixed with 1.9 μl ofGenomeWalker Adapter (25 μM), 1.6 μl 10× Ligation Buffer, and 0.5 μl T4DNA Ligase (6 units/μl). These reactions were incubated overnight at 16°C. The reactions were stopped with incubation at 70° C. for fiveminutes. After the reaction was stopped, 72 μl of TE was added to eachtube, and the contents were mixed thoroughly.

A first PCR reaction was performed using primer AP1, supplied by themanufacturer, with different primers designed within the knownheterologous insert DNA sequence (Round 1 “gene specific primers” or“GSP1”). The following reagents were mixed in a PCR tube on ice: 1 μl ofthe appropriate MIR162 DNA library, 1 μl 10 μM AP1, 1 μl 10 μM GSP1, 1μl 10 mM dNTPs, 5 μl 10× Advantage 2 PCR Buffer, 1 μl of BD Advantage 2Polymerase, and 40 μl distilled water. PCR was completed using thefollowing program: seven cycles at 94° C. for 25 seconds and 72° C. forfour minutes, 32 cycles at 94° C. for 25 seconds and 67° C. for fourminutes, and one cycle at 67° C. for four minutes. Each primary PCRreaction was diluted 50-fold by adding 1 μl of the primary PCR productwith 49 μl of distilled water. The reactions that worked were (1) theDraI and the XmnI libraries with the GSP1 primer 162GW3F1:5′TCTCTTGCTAAGCTGGGAGCTCGATCCG3′ (SEQ ID NO: 56) and primer AP1.

A second PCR reaction was performed independently using primer AP2,supplied by the manufacturer, with different primers designed within theknown heterologous insert DNA sequence (Round 2 “gene specific primers”or “GSP2”). The following reagents were mixed in a PCR tube on ice: 1 μlof the appropriate diluted primary PCR product, 1 μl 10 μM AP2, 1 μl 10μM GSP2, 1 μl 10 mM dNTPs, 5 μl 10× Advantage 2 PCR Buffer, 1 μl of BDAdvantage 2 Polymerase, and 40 μl distilled water. PCR was completedusing the following program: five cycles at 94° C. for 25 seconds and72° C. for four minutes, 20 cycles at 94° C. for 25 seconds and 67° C.for four minutes, and one cycle at 67° C. for four minutes. Thereactions that worked were (1) the DraI and the XmnI libraries with theGSP2 primer 162GW3F2: 5′AAGATTGAATCCTGTTGCCGGTCTTGCG3′ (SEQ ID NO: 57)and primer AP2.

Ten μl of the PCR products were run on a 1% agarose gel containingethidium bromide. The appropriate band was extracted from the agarosegel and purified using a Qiagen Qiaquick Gel Extraction Kit according tothe manufacturer's instructions. The extracted DNA was cloned into anInvitrogen TOPO-XL cloning vector according to the manufacturer'sinstructions. This clone was transformed into E. coli, and the plasmidDNA was extracted from the cells after overnight growth with a QiagenMiniprep kit according to the manufacturer's instructions. This plasmidwas sequenced using end run sequencing.

A new primer was designed within the new, previously unknown sequence tobe used with a primer in the insert DNA to amplify approximately 1 kb of3′ flanking sequence out of the genomic DNA. The insert DNA primer162GW3F1: 5′TCTCTTGCTAAGCTGGGAGCTCGATCCG3′ (SEQ ID NO: 56) and a 3′flanking sequence primer 1623′GWR1: 5CTGGTGAACCGATTTTTACGGAGG3′ (SEQ IDNO: 58) were used to amplify a nucleic acid molecule comprising the 3′flanking sequence for confirmation. The sequence of the resultingamplicon is set forth in SEQ ID NO: 59. This 3′ amplicon comprises the3′ junction sequence set forth in SEQ ID NO: 47. Ten μl of the PCRamplicon was run on a 1% agarose gel containing ethidium bromide. Theappropriate band was extracted from the agarose gel and purified using aQiagen Qiaquick Gel Extraction Kit according to the manufacturer'sinstructions. The extracted DNA was cloned into an Invitrogen TOPO-XLcloning vector according to the manufacturer's instructions. This clonewas transformed into E. coli. Plasmid DNA was extracted from the cellsafter overnight growth in media with a Qiagen Miniprep kit according tothe manufacturer's instructions. Three plasmids were completelysequenced using the primers shown in Table 5. The plasmid sequences werealigned to generate the complete confirmed 3′ flanking sequence (SEQ IDNO: 48).

TABLE 5 Primer sequences. Primer Name Sequence (5′→3′) Sequence No.b00106a GATTGAATCCTGTTGCC SEQ ID NO: 81 b00106b TCTCATAAATAACGTCATGC SEQID NO: 82 b00108a TCTGTGGATAACCGTATTAC SEQ ID NO: 83 b00201hTTCACGGGAGACTTTATCTG SEQ ID NO: 60 b00605a CCGATTCATTAATGCAG SEQ ID NO:61 b00704h AATAATATCACTCTGTACATCC SEQ ID NO: 64 b00712eAGTAACATAGATGACACCGC SEQ ID NO: 84 b01106a CCAGTGTGCTGGAATTCG SEQ ID NO:85 b01106f GTTGTAAAACGACGG SEQ ID NO: 65 b01107h CCAGTGTGATGGATATCTGCSEQ ID NO: 86 b01108e CCAGTGTGCTGGAATTCG SEQ ID NO: 87 b01111fCCAGTGTGATGGATATCTGC SEQ ID NO: 88 b01709f TAGGCACCCCAGGCTTTA SEQ ID NO:66 b02701a GTGTGCTGGAATTCGCCCTT SEQ ID NO: 89 b02701eTATCTGCAGAATTCGCCCTT SEQ ID NO: 90 b02702a GTGTGCTGGAATTCGCCCTT SEQ IDNO: 91 b02702e TATCTGCAGAATTCGCCCTT SEQ ID NO: 92 b02703aGTGTGCTGGAATTCGCCCTT SEQ ID NO: 93 b02703e TATCTGCAGAATTCGCCCTT SEQ IDNO: 94 b02704a GTGTGCTGGAATTCGCCCTT SEQ ID NO: 95 b02811aGGTCTTGCGATGATTATC SEQ ID NO: 96 b05104c GAGAGGAATGGCAGCAGA SEQ ID NO:97 b05104d CATGACGGGTTTGAGATT SEQ ID NO: 98 b05104e AATCTCAAACCCGTCATGSEQ ID NO: 99 b05104f TCTGCTGCCATTCCTCTC SEQ ID NO: 100 b05104gGATCAACCCGGAGAGGAAT SEQ ID NO: 101 b05104h CCATGACGGGTTTGAGAT SEQ ID NO:102 b05105c CAACCGACCTGACAAGTGAC SEQ ID NO: 103 b05105eATCTCAAACCCGTCATGG SEQ ID NO: 104 b05105f ATTCCTCTCCGGGTTGATC SEQ ID NO:105

Example 6. Detection of Vip3Aa20 Protein in MIR162 by ELISA

Extracts were prepared from MIR162 leaves, roots, pith, kernels, silk,pollen and whole plants. They were quantitatively analyzed for Vip3Aa20by ELISA using immunoaffinity purified goat anti-Vip3A and ProteinA-purified rabbit anti-Vip3A polyclonal antibodies using art recognizedELISA procedures. Vip3Aa20 was detected in all tissues analyzed acrossall growth stages. The mean level of Vip3Aa20 protein detected in thewhole plant at anthesis and seed maturity was 10 μg/g fresh weight and16 μg/g fresh weight, respectively. The mean level of Vip3Aa20 proteinin leaves at anthesis was 22 μg/g fresh weight.

Example 7. Field Efficacy of MIR162

The MIR162 event was tested in the field for efficacy against fallarmyworm (FAW, Spodoptera frugiperda), corn earworm (CEW, Helicoverpazea), black cutworm (BCW, Agrotis ipsilon), and European corn borer(ECB, Ostrinia nubilalis). Performance of the MIR162 event was comparedwith that of Warrior (Syngenta, Inc.), a conventional insecticidestandard applied at a rate of 11.2 g a.i./acre, the transgenic cornevent Bt11, comprising a cry1Ab gene, and a Bt11×MIR162 hybrid, producedby crossing a Bt11 inbred line with a MIR162 inbred line.

Twenty-eight trials were planted in 13 states that represented the majorcorn growing regions of the continental United States. Trials wereplanted in a randomized complete block design with four replicated plotsper block. Plots were 17.5 row feet per treatment per replication.Planting density was targeted at approximately 30,000 plants/acre.Immuno-diagnostic strips were used to confirm the presence or absence ofthe Vip3Aa20 and Cry1Ab proteins in the different treatment groups.

Natural pest infestations were utilized in trials where populations weresufficiently high; where they were not, artificial infestations werecarried out. Artificial infestation with two 2^(nd)- to 3^(rd)-instarlarvae at V1-V2 was utilized in the BCW trials. Plots were rated at 3,7, and 14 days post-infestation. BCW damage was recorded as partiallydamaged plants and fully cut plants. FAW plots were rated 7 and 14 dayspost-infestation or after 3^(rd)-instar larvae were observed in controlplants. The following scale was used to evaluate FAW and CEW leafdamage:

-   -   0.01—No visible leaf damage    -   1—Pin-hole damage on a few leaves    -   2—Small amount of shot-hole damage on a few leaves    -   3—Shot-hole damage on several leaves    -   4—Shot-hole damage and lesions on a few leaves    -   5—Lesions on several leaves    -   6—Large lesions on several leaves    -   7—Large lesions and portions eaten away on a few leaves    -   8—Large lesions and portions eaten away on several leaves    -   9—Large lesions and portions eaten away on most leaves

Plant damage was assessed for both first and second generation ECB. Thefollowing scale was used for rating first generation damage, typicallywhen larvae were in the 3^(rd) to 4th instar:

-   -   1—No visible leaf damage    -   2—Small amount of shot-hole injury on a few leaves    -   3-Shot-hole injury common on several leaves    -   4—Several leaves with shot-holes and elongated lesions    -   5—Several leaves with elongated lesions    -   6—Several leaves with elongated lesions about 2.5 cm    -   7—Long lesions common on about one half of the leaves    -   8—Long lesions common on about two-thirds of the leaves    -   9—Most leaves with long lesions

Second generation ECB damage was assessed three to four weeks afterartificial infestation or the end of the peak egg laying period. Thefollowing measurements were taken: number live larvae/stalk, number livelarvae/shank, number live larvae/ear, number of tunnels/stalk,cumulative tunnel length (cm)/stalk, cumulative tunnel length(cm)/shank, number tunnels/ear, cumulative tunnel length kernel damage(cm)/ear, and % infested plants.

CEW trials were generally planted late to increase natural infestationlevels. Feeding damage to ears was evaluated when CEW larvae on controlplants were at the L5-L6 growth stage. Ear ratings included recordingthe number of larvae observed per ear and length of visible kernelfeeding measured from the ear tip to the average lowest kerneldestroyed.

Results of the BCW field trial are shown in Table 6. Less than 3% of theMIR162 plants and Bt11×MIR162 plants were cut by BCW larvae. Significantnumbers of Bt11 and control plants were cut. Plants comprising theMIR162 genotype had less BCW feeding damage than the conventionalinsecticide treated plants.

TABLE 6 Stalk damage ratings from five trials with BCW at 21 days afterinfestation. Damage was measured as percent of total plants cut.Treatment % Cut Plants MIR162 2 Bt11 42 Bt11 X MIR162 3 WarriorInsecticide 12 Negative Control 40

The FAW field trial results are shown in Table 7. FAW feeding damage wasmeasured on a scale of 0.01 to 9. Mean feeding damage in the MIR162hybrids was very low (<1) and significantly lower than average damageobserved in the Bt11 and conventional insecticide treatments. Insectpressure in these trials was heavy with approximately 50 to 100 neonatelarvae/plant. Bt11 provided some protection from damage, whereas theconventional insecticide treatment provided no protection, sustainingthe same amount of damage as the control.

TABLE 7 Leaf feeding damage ratings from five trials for FAW. Meandamage ratings at 14 days after infestation are presented for eachtreatment. Treatment Mean Leaf Damage Rating (0.01-9) MIR162 0.90 Bt112.52 Bt11 X MIR162 0.84 Warrior Insecticide 3.60 Negative Control 3.78

Results of the trials to assess first generation ECB damage arepresented in Table 8. ECB feeding damage was rated on a scale of 1-9. Inthese trials, MIR162 conferred minimal protection against ECB feedingdamage. Bt11 fully protected the plants from ECB feeding damage. TheBt11×MIR162 plants had the same level of protection as the Bt11 plants.The conventional insecticide treatment provided better protection thanthe MIR162 trait but significantly less protection than that provided byBt11.

TABLE 8 Leaf feeding damage field trial. Mean damage ratings at 14 daysafter infestation. Treatment Mean Leaf Damage Rating (1-9) MIR162 2.95Bt11 1.00 Bt11 X MIR162 1.00 Warrior Insecticide 2.05 Negative Control3.88

Second generation ECB damage results are presented in Table 9. Feedingdamaged was measured as cumulative tunnel length in each corn stalk (ifmore than one tunnel was found, tunnel lengths were summed). The Bt11and Bt11×MIR162 treatments provided strong protection against stalkboring, whereas no protection against tunneling was provided by MIR162alone or the insecticide treatment.

TABLE 9 Stalk damage ratings from seven trials for second generation ECBlarvae measured in tunnel length (cm) per stalk. Measurements were takenthree to four weeks after artificial infestation. Treatment Mean TunnelLength (cm) MIR162 5.46 Bt11 0.37 Bt11 X MIR162 0.48 Warrior Insecticide5.06 Negative Control 5.04

Results of trials to assess CEW damage are presented in Table 10.Feeding damage was rated as length of kernel damage per ear, measuredfrom the ear tip to the average lowest kernel destroyed. Significant eardamage was observed in the Bt11, insecticide, and check plots. Bt11provided some level of protection compared to untreated check and wascomparable to the protection provided by the conventional insecticidetreatment. MIR162 and Bt11×MIR162 provided almost complete protection ofthe ears from CEW larval feeding damage.

TABLE 10 Ear damage ratings from six trials for CEW measured as averagelength of feeding damage. Measurements were taken when CEW larvae wereL5-L6 in check plants. Treatment Mean Ear Damage (cm) MIR162 0.17 Bt112.24 Bt11 X MIR162 0.02 Warrior Insecticide 2.20 Negative Control 3.42

Example 8. Efficacy of MIR162 Against Western Bean Cutworm

Current commercial transgenic events producing Cry1Ab protein have notprovided acceptable levels of protection against the western beancutworm (WBCW, Striacosta albicosta). Therefore, MIR162 alone andstacked with other transgenic genotypes was tested for efficacy againstWBCW.

WBCW eggs were collected from wild caught female moths. Larvae were fedon a meridic black cutworm diet until use in the experiments. Cornplants were field grown.

The following treatments were tested: MIR162, Bt11, MIR604, MIR162×Bt11,MIR162×MIR604, MIR604×Bt11, Force® (Syngenta, Inc.), a conventionalinsecticide applied at planting to a negative isoline, and two negativecontrol isolines. MIR604 is a novel transgenic corn event that comprisesa cry3A055 gene encoding a protein that is active against corn rootworm(Diabrotica spp.) larvae and is disclosed in US Patent Applicationpublication No. 2005/0216970, published Sep. 29, 2005, hereinincorporated by reference.

For the experiments, a two-inch piece of green silks and husk was cutfrom ears from field grown corn plants in each treatment andreplication. The terminal brown ends of the silks were removed and thehusk discarded. Approximately 1.5 inches of silks were placed inindividual 14 ml plastic cups. One larva was then placed in each cup andthe cups sealed. Several different stages of larvae were tested rangingfrom 3^(rd) to 6^(th)-instars. Cups containing silks and larvae wereheld at natural day length and room temperature for the duration of theexperiments. Larval survival was recorded after eight days. Treatmentswere replicated four times per experiment.

Results of the WBCW experiments are presented in Table 11. Survival ofWBCW on silks from the negative isolines and the conventionalinsecticide treatment were nearly 100%. Survival of WBCW larvae on Bt11and MIR604 silks, tested either alone or in combination in the sameplant, was not different from survival on the negative isolines.Survival of WBCW larvae was reduced when larvae were fed silks fromMIR162. The combination of MIR162×Bt11 in the same plant did notdecrease survival any further than MIR162 alone. However, surprisingly,when the MIR162 transgenic genotype was stacked with the MIR604transgenic genotype in the same plant, larval mortality significantlyincreased compared to MIR162 or MIR604 alone.

TABLE 11 Percent (±SE) survival of WBCW larvae on corn silks. ExperimentNumber Treatment 1 2 3 4 5 6 7 Bt11 75 (25) 75 (25) 100 (0) 100 (0) 100(0)  100 (0)  100 (0) MIR162 25 (25) 0 (0)  0 (0)  50 (29) 50 (29) 50(29)  0 (0) MIR604 100 (0)  100 (0)  100 (0) MIR162xBt11 25 (25) 25 (25) 0 (0)  0 (0) 25 (25) 25 (25)  25 (25) MIR162xMIR604 0 (0) 25 (25)  50(29) MIR604xBt11  75 (25) Force 100 (0)  100 (0)  100 (0) Neg. Control#1 100 (0)  75 (25) 100 (0) 100 (0) 75 (25) 100 (0)  100 (0) Neg.Control #2 100 (0)  100 (0)  100 (0) 100 (0) 100 (0)  100 (0)  100 (0)

Example 9. Use of Event Mir162 Insertion Site for Targeted Integrationin Maize

The MIR162 flanking sequences disclosed in SEQ ID NO: 46 and SEQ ID NO:48 were used to search maize genomic databases. Identical matches toboth flanking sequences where found on a BAC clone, CH201-307P5, ofchromosome 5 (NCBI Accession No. AC185313) on Contig 13 (SEQ ID NO:106). More specifically, the MIR162 insert is on chromosome 5 between a5′ molecular marker, designated herein as the Opie2 marker (nucleotides1680-3338 of SEQ ID NO: 106), and a 3′ molecular marker, designatedherein as the gag marker (nucleotides 43,275-45,086 of SEQ ID NO: 106).Using this information, it was determined that the heterologous DNAinserted into MIR162 displaced 58 nucleotides of maize genomic DNA,nucleotides 25,455 to 25,512 of SEQ ID NO: 106 (also shown as SEQ ID NO:107), which is between the 5′ flanking sequence (nucleotides 1-25,454 ofSEQ ID NO: 106) and the 3′ flanking sequence (nucleotides 25,513-51,328of SEQ ID NO: 106).

Consistent agronomic performance of the transgene of event MIR162 overseveral generations under field conditions suggests that theseidentified regions around the MIR162 insertion site provide good genomiclocations for the targeted integration of other transgenic genes ofinterest. Such targeted integration overcomes the problems withso-called “positions effects,” and the risk of creating a mutation inthe genome upon integration of the transgene into the host. Furtheradvantages of such targeted integration include, but are not limited to,reducing the large number of transformation events that must be screenedand tested before obtaining a transgenic plant that exhibits the desiredlevel of transgene expression without also exhibiting abnormalitiesresulting from the inadvertent insertion of the transgene into animportant locus in the host genome. Moreover, such targeted integrationallows for stacking transgenes rendering the breeding of elite plantlines with both genes more efficient.

Using the above disclosed teaching, the skilled person is able to usemethods know in the art to target heterologous nucleic acids of interestto the same insertion site on chromosome 5 as that in MIR162 or to asite in close proximity to the insertion site in MIR162. One such methodis disclosed in US Patent Application Publication No. 20060253918,herein incorporated by reference in its entirety. Briefly, up to 20 Kbof the genomic sequence flanking 5′ to the insertion site (nucleotides5,454 to 25,454 of SEQ ID NO: 106) and up to 20 Kb of the genomicsequence flanking 3′ to the insertion site (nucleotides 25,513 to 45,513of SEQ OD NO: 106) are used to flank the gene or genes of interest thatare intended to be inserted into a genomic location on Chromosome 5 viahomologous recombination. These sequences can be further flanked byT-DNA border repeats such as the left border (LB) and right border (RB)repeat sequences and other booster sequences for enhancing T-DNAdelivery efficiency. The gene or genes of interest can be placed exactlyas in the MIR162 insertion site or can be placed anywhere within the 20Kb regions around the MIR162 insertion sites to confer consistent levelof transgene expression without detrimental effects on the plant. TheDNA vectors containing the gene or genes of interest and flankingsequences can be delivered into plant cells via one of the severalmethods known to those skilled in the art, including but not limited toAgrobacterium-mediated transformation. The insertion of the DNA vectorinto the MIR162 target site can be further enhanced by one of theseveral methods, including but not limited to the co-expression orup-regulation of recombination enhancing genes or down-regulation ofendogenous recombination suppression genes. Furthermore, it is known inthe art that cleavage of specific sequences in the genome can be used toincrease homologous recombination frequency, therefore insertion intothe MIR162 insertion site and its flanking regions can be enhanced byexpression of natural or designed sequence-specific endonucleases forcleaving these sequences. Thus, using the teaching provided herein, anyheterologous nucleic acid can be inserted on maize chromosome 5 at atarget site located between nucleotides 25,454 and 45,513 of SEQ ID NO:106 or a target site in the vicinity to this site.

Example 10. Use of Event MIR162 Insertion Site and Flanking Sequencesfor Stabilization of Gene Expression

The genomic sequences flanking the MIR162 insertion site may also beused to stabilize expression of other gene(s) of interest when insertedas a transgene in other genomic locations in maize and other crops.Specifically, up to 20 Kb of the genomic sequence flanking 5′ to theinsertion site (nucleotides 5,454 to 25,454 of SEQ ID NO: 106) and up to20 Kb of the genomic sequence flanking 3′ to the insertion site(nucleotides 25,513 to 45,513 of SEQ OD NO: 106) are used to flank thegene or genes of interest that are intended to be inserted into thegenome of plants. These sequences can be further flanked by T-DNA borderrepeats such as the left border (LB) and right border (RB) repeatsequences and other booster sequences for enhancing T-DNA deliveryefficiency. The gene or genes of interest can be placed exactly as inthe MIR162 insertion site or can be placed anywhere within the 20 Kbregions around the MIR162 insertion sites to confer consistent level oftransgene expression. The DNA vectors containing the gene or genes ofinterest and MIR162 insertion site flanking sequence can be deliveredinto plant cells via one of the several methods known to those skilledin the art, including but not limited to protoplast transformation,biolistic bombardment and Agrobacterium-mediated transformation. Thedelivered DNA can be integrated randomly into a plant genome or can alsobe present as part of the independently segregating genetic units suchas artificial chromosome or mini-chromosome. The DNA vectors containingthe gene(s) of interest and the MIR162 insertion site flanking sequencescan be delivered into plant cells. Thus, by surrounding a gene or genesof interest with the genomic sequence flanking the MIR162 insertionsite, the expression of such genes are stabilized in a transgenic hostplant such as a dicot plant or a monocot plant like corn.

DEPOSIT

Applicants have made a deposit of corn seed of event MIR162 disclosedabove on 23 Jan. 2007 in accordance with the Budapest Treaty at theAmerican Type Culture Collection (ATCC), 1801 University Boulevard,Manassas, Va. 20110 under ATCC Accession No. PTA-8166. The deposit willbe maintained in the depositary for a period of 30 years, or 5 yearsafter the last request, or the effective life of the patent, whicheveris longer, and will be replaced as necessary during that period.Applicants impose no restrictions on the availability of the depositedmaterial from the ATCC; however, Applicants have no authority to waiveany restrictions imposed by law on the transfer of biological materialor its transportation in commerce. Applicants do not waive anyinfringement of their rights granted under this patent or under thePlant Variety Protection Act (7 USC 2321 et seq.).

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent document was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A method of detecting the presence of a nucleicacid molecule that is unique to event MIR162 in a sample comprising cornnucleic acids, the method comprising: (a) contacting the sample with apair of primers that, when used in a nucleic-acid amplification reactionwith genomic DNA from event MIR162 produces an amplicon that isdiagnostic for event MIR162; (b) performing a nucleic acid amplificationreaction, thereby producing the amplicon; and (c) detecting theamplicon.
 2. A method of detecting the presence of a nucleic acidmolecule that is unique to event MIR162 in a sample comprising cornnucleic acids, the method comprising: (a) contacting the sample with aprobe that hybridizes under high stringency conditions with genomic DNAfrom event MIR162 and does not hybridize under high stringencyconditions with DNA of a control corn plant, wherein the probe comprisesthe nucleotide sequence of SEQ ID NO: 45 or SEQ ID NO: 47; (b)subjecting the sample and probe to high stringency hybridizationconditions; and (c) detecting hybridization of the probe to the nucleicacid molecule.
 3. An amplicon diagnostic for event MIR162, wherein saidamplicon is at least 20 nucleotides in length and comprises the nucleicacid sequence of SEQ ID NO: 45, SEQ ID NO: 47, the nucleotide ofposition 387 of SEQ ID NO: 1 or the nucleotide of position 1683 of SEQID NO:
 1. 4. A pair of polynucleotide primers comprising a firstpolynucleotide primer and a second polynucleotide primer which functiontogether in the presence of an event MIR162 DNA template in a sample toproduce an amplicon diagnostic for event MIR162, wherein said firstprimer and second primer comprise at least 10 contiguous nucleotides ofSEQ ID NO: 1 and wherein said amplicon is at least 20 nucleotides inlength and comprises the nucleotide of position 387 of SEQ ID NO: 1and/or position 1683 of SEQ ID NO:
 1. 5. A method of detecting thepresence of a nucleic acid molecule that is unique to event MIR162 in asample comprising corn nucleic acids, the method comprising: (a)contacting the sample with a pair of primers of claim 4 that, when usedin a nucleic-acid amplification reaction with genomic DNA from eventMIR162 produces an amplicon that is diagnostic for event MIR162; (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon.